MF研究者総覧

研究者データベース

詳細
出村　誠(デムラ　マコト)
先端生命科学研究院　生命機能科学研究部門　生物情報解析科学研究分野
教授
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Last Updated :2023/12/09

基本情報

所属

  • 先端生命科学研究院　生命機能科学研究部門　生物情報解析科学研究分野

職名

  • 教授

学位

  • 薬学博士（北海道大学）

ホームページURL

J-Global ID

研究キーワード

  • SDGs   ソフトマター   バイオミミクリー   ロドプシン   絹   タンパク質   NMR   生物物理学   Protein Science (4708)   NMR (5801)   Biophysics (5803)   

研究分野

  • ナノテク・材料 / 高分子材料
  • ライフサイエンス / 構造生物化学
  • ライフサイエンス / 生物物理学

担当教育組織

職歴

  • 2022年04月 - 現在 北海道大学 創成研究機構 研究人材育成推進室長
  • 2021年04月 - 現在 北海道大学 総長補佐 (サステイナビリティ推進機構, 研究戦略室兼務)
  • 2006年04月 - 現在 北海道大学 大学院先端生命科学研究院 教授
  • 2019年04月 - 2023年03月 北海道大学 大学院先端生命科学研究院 副研究院長
  • 2020年04月 - 2022年03月 北海道大学 人材育成本部 副本部長
  • 2009年 - 2021年 北海道大学 人材育成本部(兼務)
  • 2013年04月 - 2019年03月 北海道大学 大学院先端生命科学研究院 研究院長
  • 2006年 - 2016年 大阪大学蛋白質研究所 共同研究員
  • 2010年04月 - 2013年03月 北海道大学 大学院先端生命科学研究院 副研究院長
  • 1998年10月 - 2006年03月 北海道大学 大学院理学研究科 助教授
  • 2004年 筑波大学 非常勤講師
  • 1992年01月 - 1998年09月 東京農工大学機器分析センター 専任講師
  • 1997年 北海道大学 非常勤講師
  • 1986年04月 - 1992年01月 東京農工大学工学部物質生物工学科 教務職員

学歴

  • 1983年04月 - 1986年03月   北海道大学   大学院薬学研究科製薬化学専攻博士課程修了(薬学博士)

所属学協会

  • 日本核磁気共鳴学会   日本蛋白質科学会   高分子学会   繊維学会   日本化学会   日本生物物理学会   日本蚕糸学会   

研究活動情報

論文

  • Yukino Sato, Tsubasa Hashimoto, Koji Kato, Akiko Okamura, Kaito Hasegawa, Tsukasa Shinone, Yoshikazu Tanaka, Yoshiki Tanaka, Tomoya Tsukazaki, Takashi Tsukamoto, Makoto Demura, Min Yao, Takashi Kikukawa
    The Journal of biological chemistry 105393 - 105393 2023年10月25日 [査読有り]
     
    Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin (NaR), which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of NaR from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.
  • Yuya Ohki, Tsukasa Shinone, Sayo Inoko, Miu Sudo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
    The Journal of biological chemistry 105305 - 105305 2023年09月29日 [査読有り]
     
    Previous research of anion channelrhodopsins (ACRs) has been performed using cytoplasmic domain (CPD)-deleted constructs, and therefore have overlooked the native functions of full-length ACRs and the potential functional role(s) of the CPD. In this study, we used the recombinant expression of full-length Guillardia theta ACR1 (GtACR1_full) for pH measurements in Pichia pastoris cell suspensions as an indirect method to assess its anion transport activity, and for absorption spectroscopy and flash photolysis characterization of the purified protein. The results show that the CPD, which was predicted to be intrinsically disordered and possibly phosphorylated, enhanced NO3- transport compared to Cl- transport, which resulted in the preferential transport of NO3-. This correlated with the extended lifetime and large accumulation of the photocycle intermediate that is involved in the gate-open state. Considering that the depletion of a nitrogen source enhances the expression of GtACR1 in native algal cells, we suggest that NO3- transport could be the natural function of GtACR1_full in algal cells.
  • Li Gan, Yuga Inamura, Yu Shimizu, Yuki Yokoi, Yuki Ohnishi, Zihao Song, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Masaaki Ito, Tokiyoshi Ayabe, Kiminori Nakamura, Tomoyasu Aizawa
    Metabolites 13 9 1003 - 1003 2023年09月10日 [査読有り]
     
    Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01–0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.
  • Yuhei Doi, Jo Watanabe, Ryota Nii, Takashi Tsukamoto, Makoto Demura, Yuki Sudo, Takashi Kikukawa
    Scientific reports 12 1 16422 - 16422 2022年09月30日 [査読有り]
     
    Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42-, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42- pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42- transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42- pump activity. Notably, the MrHR and SyHR mutants did not show SO42- induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42- in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42- into their centers after illumination and release it before the ends of the photoreactions.
  • Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa
    ACS Omega 2022年09月02日 [査読有り]
  • Yuji Sato, Yi Wang, Yuchi Song, Weiming Geng, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Makoto Demura, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Amino acids 2022年01月17日 [査読有り]
     
    Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.
  • Syogo Sasaki, Jun Tamogami, Koki Nishiya, Makoto Demura, Takashi Kikukawa
    The Journal of biological chemistry 297 3 101013 - 101013 2021年09月 [査読有り]
     
    Many H+-pump rhodopsins conserve "H+ donor" residues in cytoplasmic (CP) half channels to quickly transport H+ from the CP medium to Schiff bases at the center of these proteins. For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the medium, whereas the Lys donor first captures H+ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer reactions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.
  • Tomoya Kato, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
    The Journal of biological chemistry 100792 - 100792 2021年05月18日 [査読有り]
     
    Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters due to the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+ pump rhodopsin (NaR). We triggered the transport cycle of NaR using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions, and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.
  • Chihiro Kikuchi, Hina Kurane, Takuma Watanabe, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
    Scientific reports 11 1 7908 - 7908 2021年04月12日 [査読有り]
     
    Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO3- of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO3- transport and might take part in NO3--related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3rd transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO3-. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.
  • Keisuke Murabe, Takashi Tsukamoto, Tomoyasu Aizawa, Makoto Demura, Takashi Kikukawa
    Journal of the American Chemical Society 142 37 16023 - 16030 2020年09月16日 [査読有り][通常論文]
     
    For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle.
  • Ayumi Yamamoto, Takashi Tsukamoto, Kenshiro Suzuki, Eri Hashimoto, Yoshihiro Kobashigawa, Kousuke Shibasaki, Takeshi Uchida, Fuyuhiko Inagaki, Makoto Demura, Koichiro Ishimori
    Biophysical journal 118 11 2853 - 2865 2020年06月02日 [査読有り][通常論文]
     
    We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.
  • Tomoyasu Aizawa, Makoto Demura, Kazutoshi Gohara, Hisashi Haga, Koichiro Ishimori, Masataka Kinjo, Tamiki Komatsuzaki, Katsumi Maenaka, Min Yao
    Biophysical reviews 12 2 233 - 236 2020年04月 [査読有り][通常論文]
  • Azusa Iizuka, Kousuke Kajimoto, Tomotsumi Fujisawa, Takashi Tsukamoto, Tomoyasu Aizawa, Naoki Kamo, Kwang-Hwan Jung, Masashi Unno, Makoto Demura, Takashi Kikukawa
    Scientific reports 9 1 10711 - 10711 2019年07月24日 [査読有り][通常論文]
     
    Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the "monomerization effects": Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.
  • 堀江裕紀子, 根本英幸, 藤田 仁, 池川繁男, 熊木康裕, 大西裕季, 久米田博之, 出村 誠, 相沢智康
    日本食品科学工学会誌 66 4 139 - 146 2019年04月15日 [査読有り]
  • Takatoshi Hasemi, Takashi Kikukawa, Yumi Watanabe, Tomoyasu Aizawa, Seiji Miyauchi, Naoki Kamo, Makoto Demura
    Biochimica et biophysica acta. Bioenergetics 1860 2 136 - 146 2019年02月01日 [査読有り][通常論文]
     
    Mastigocladopsis repens halorhodopsin (MrHR) is a Cl--pumping rhodopsin that belongs to a distinct cluster far from other Cl- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I- similar to Cl- but cannot transport it. I--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl--pumping photocycle, Cl- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl--binding affinity nor the absorption spectrum, but completely eliminated the Cl--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H+ release was detected for photolyzed MrHR by using an indium‑tin oxide electrode, which acts as a good time-resolved pH sensor. This H+ release disappeared in the I--bound form of the wild-type and Cl--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl- movement to the CP channel.
  • Takashi Tsukamoto, Chihiro Kikuchi, Hiromu Suzuki, Tomoyasu Aizawa, Takashi Kikukawa, Makoto Demura
    Scientific reports 8 1 13445 - 13445 2018年09月07日 [査読有り][通常論文]
     
    Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl- dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl- in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl- concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl- non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl- conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl-.
  • Jun Tamogami, Takashi Kikukawa, Keisuke Ohkawa, Noboru Ohsawa, Toshifumi Nara, Makoto Demura, Seiji Miyauchi, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama, Kazumi Shimono, Naoki Kamo
    Journal of Photochemistry and Photobiology B: Biology 183 35 - 45 2018年06月01日 [査読有り][通常論文]
     
    Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.
  • Yu Nakajima, Takashi Tsukamoto, Yohei Kumagai, Yoshitoshi Ogura, Tetsuya Hayashi, Jaeho Song, Takashi Kikukawa, Makoto Demura, Kazuhiro Kogure, Yuki Sudo, Susumu Yoshizawa
    Microbes and environments 33 1 89 - 97 2018年03月29日 [査読有り][通常論文]
     
    Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl--pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl- pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl--transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl--pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.
  • Dai G, Geng X, Chao L, Tamogami J, Kikukawa T, Demura M, Kamo N, Iwasa T
    Photochemistry and photobiology 2018年03月 [査読有り][通常論文]
  • Md. Ruhul Kuddus, Megumi Yamano, Farhana Rumi, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
    BIOTECHNOLOGY PROGRESS 33 6 1520 - 1528 2017年11月 [査読有り][通常論文]
     
    Snakin-1 (SN-1) is a cysteine-rich plant antimicrobial peptide and the first purified member of the snakin family. SN-1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN-1 in Escherichia coli by a previously developed coexpression method using an aggregation-prone partner protein. Our goal was to increase the productivity of SN-1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN-1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN-1, the identity of which was verified by MALDI-TOF MS and NMR studies. The purified recombinant SN-1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation-prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN-1. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520-1528, 2017
  • Kousuke Kajimoto, Takashi Kikukawa, Hiroki Nakashima, Haruki Yamaryo, Yuta Saito, Tomotsumi Fujisawa, Makoto Demura, Masashi Unno
    JOURNAL OF PHYSICAL CHEMISTRY B 121 17 4431 - 4437 2017年05月 [査読有り][通常論文]
     
    Sodium-ion-pump rhodopsin (NaR) is a microbial rhodopsin that transports Na+ during its photocycle. Here we explore the photocycle mechanism of NaR from Indibacter alkaliphilus with transient absorption and transient resonance Raman spectroscopy. The transient absorption data indicate that the photocycle of NaR is K (545 nm) -> L (490 nm)/M (420 mu) O-1 (590 rim) O-2 (56Q mu) NaR, where the L and M are formed as equilibrium states. The presence of K, L, M, and O intermediates was confirmed by the resonance Raman spectra with 442 and 532 nm excitation. The main component of the transient resonance Raman spectra was due to L which contains a 1'3 -cis retinal protonated Schiff base. The presence of an enhanced hydrogen out -of -plane band, as well as its sensitivity to the H/D exchange indicate that the retinal chromophore is distorted near the Schiff base region in L. Moreover, the retinal Schiff base of the L state forms a hydrogen bond That is stronger than that of the dark state. These observations are consistent with a Na+ pumping mechanism that involves a proton transfer from the retinal Schiff base to a key aspartate residue (Asp116 in Krokinobacter eikastus rhodopsin 2) in the L/M states.
  • Takashi Tsukamoto, Susumu Yoshizawa, Takashi Kikukawa, Makoto Demura, Yuki Sudo
    JOURNAL OF PHYSICAL CHEMISTRY B 121 9 2027 - 2038 2017年03月 [査読有り][通常論文]
     
    Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called "NTQ' rhodopsins, contain a conserved Asn-Thr-Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08(T) strain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl-in the-vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl-to 534 nm in the presence of Cl-. From the Cl- concentration dependence, we estimated the affinity (dissociation constant, K-d) for Cl- in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl-pumping rhodopsin (C1R). NM-R3 showed no dark-light adaptation of the retinal chromophore and predominantly possessed an all-trans-retinal, which is responsible for the light-driven Cl-pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L(N), O-1, O-2, and NM-R3'). From these results, we assume that the Cl-is released and taken up during the L(N)-O-1 transition from a transiently formed cytoplasmic (CP) binding site and the O-2-NM R3' or the NM-R3'-original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl-transport by NM-R3 based on our results and its recently reported crystal structure.
  • Tamogami J, Kikukawa T, Nara T, Demura M, Kimura-Someya T, Shirouzu M, Yokoyama S, Miyauchi S, Shimono K, Kamo N
    Biophysics and physicobiology 14 49 - 55 2017年 [査読有り][通常論文]
     
    A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M⇄N⇄O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.
  • Shintaro Nakamura, Takashi Kikukawa, Jun Tamogami, Masakatsu Kamiya, Tomoyasu Aizawa, Martin W. Hahn, Kunio Ihara, Naoki Kamo, Makoto Demura
    BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1857 12 1900 - 1908 2016年12月 [査読有り][通常論文]
     
    Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed lambda(max) at 534 nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P-620, could be assigned to the 0 intermediate in other H+ pumps. In contrast to conventional 0, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH 7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P-650, while the other first releases H+ prior to H+ uptake during P-620 formation. (C) 2016 Elsevier B.V. All rights reserved.
  • Nakamura S, Kikukawa T, Tamogami J, Kamiya M, Aizawa T, Hahn MW, Ihara K, Kamo N, Demura M
    Biochimica et biophysica acta 1857 12 1900 - 1908 2016年12月 [査読有り][通常論文]
  • Md. Ruhul Kuddus, Farhana Rumi, Motosuke Tsutsumi, Rika Takahashi, Megumi Yamano, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
    PROTEIN EXPRESSION AND PURIFICATION 122 15 - 22 2016年06月 [査読有り][通常論文]
     
    Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and H-1 NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. (C) 2016 Elsevier Inc. All rights reserved.
  • Mi-Hwa Baek, Masakatsu Kamiya, Takahiro Kushibiki, Taichi Nakazumi, Satoshi Tomisawa, Chiharu Abe, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
    JOURNAL OF PEPTIDE SCIENCE 22 4 214 - 221 2016年04月 [査読有り][通常論文]
     
    Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an -helical structure in a solution containing LPS. For NMR experiments, we expressed N-15-labeled and C-13-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed N-15-edited and C-13-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined -helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.
  • Motosuke Tsutsumi, Hideki Muto, Shohei Myoba, Mai Kimoto, Akira Kitamura, Masakatsu Kamiya, Takashi Kikukawa, Shigeharu Takiya, Makoto Demura, Keiichi Kawano, Masataka Kinjo, Tomoyasu Aizawa
    FEBS OPEN BIO 6 2 106 - 125 2016年02月 [査読有り][通常論文]
     
    Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and (c) two types of transient interactions between FMBP-1 and chromosomal DNA, and (d) stable binding of FMBP-1 to chromosomal DNA.
  • Tamogami J, Sato K, Kurokawa S, Yamada T, Nara T, Demura M, Miyauchi S, Kikukawa T, Muneyuki E, Kamo N
    Biochemistry 55 7 1036 - 1048 2016年02月 [査読有り][通常論文]
     
    Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H+-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as M-a), which is different from M, with a pK(a) of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pK(a) value of ca. 9.5 was estimated for this inversion and was in good agreement with the pK(a) value of the formation of M-a (similar to 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.
  • Takatoshi Hasemi, Takashi Kikukawa, Naoki Kamo, Makoto Demura
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 1 355 - 362 2016年01月 [査読有り][通常論文]
     
    Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H+ and Na+ pumps and an inward Cl- pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H+ pump bacteriorhodopsin, which was converted to a Cl- pump in 1995 by a single amino acid replacement. In this study we report the first success of the reverse conversion from a Cl- pump to a H+ pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl- pump and belongs to a cluster that is far distant from the known Cl- pumps. With a single amino acid replacement, MrHR is converted to a H+ pump in which dissociable residues function almost completely in the H+ relay reactions. MrHR most likely evolved from a H+ pump, but it has not yet been highly optimized into a mature Cl- pump.
  • Tamaki, Hajime, Egawa, Ayako, Kido, Kouki, Kameda, Tomoshi, Kamiya, Masakatsu, Kikukawa, Takashi, Aizawa, Tomoyasu, Fujiwara, Toshimichi, Demura, Makoto
    JOURNAL OF BIOMOLECULAR NMR 64 1 87 - 101 2016年01月 [査読有り][通常論文]
     
    Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a C alpha RMSD of 1.49 angstrom relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn2+ mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.
  • Munenori Furuse, Jun Tamogami, Toshiaki Hosaka, Takashi Kikukawa, Naoko Shinya, Masakatsu Hato, Noboru Ohsawa, So Young Kim, Kwang-Hwan Jung, Makoto Demura, Seiji Miyauchi, Naoki Kamo, Kazumi Shimono, Tomomi Kimura-Someya, Shigeyuki Yokoyama, Mikako Shirouzu
    ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 71 2203 - 2216 2015年11月 [査読有り][通常論文]
     
    Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 angstrom) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pK(a) of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pK(a) of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.
  • Toyoyuki Ose, Azusa Oikawa, Yukiko Nakamura, Katsumi Maenaka, Yuya Higuchi, Yuki Satoh, Shiho Fujiwara, Makoto Demura, Teruo Sone, Masakatsu Kamiya
    JOURNAL OF BIOMOLECULAR NMR 63 2 229 - 235 2015年10月 [査読有り][通常論文]
  • Takashi Kikukawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Kunio Ihara, Naoki Kamo, Makoto Demura
    BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1847 8 748 - 758 2015年08月 [査読有り][通常論文]
     
    Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR'. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR' still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR' transitions. (C) 2015 Elsevier B.V. All rights reserved.
  • Kikukawa T, Kusakabe C, Kokubo A, Tsukamoto T, Kamiya M, Aizawa T, Ihara K, Kamo N, Demura M
    Biochimica et biophysica acta 1847 8 748 - 758 2015年08月 [査読有り][通常論文]
  • Tomisawa S, Sato Y, Kamiya M, Kumaki Y, Kikukawa T, Kawano K, Demura M, Nakamura K, Ayabe T, Aizawa T
    Protein expression and purification 112 21 - 28 2015年08月 [査読有り][通常論文]
     
    Mammalian alpha-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of alpha-defensins, large amounts of alpha-defensins are essential. Although many expression systems for the production of recombinant alpha-defensins have been developed, attempts to obtain large amounts of alpha-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse alpha-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of alpha-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step. (C) 2015 Elsevier Inc. All rights reserved.
  • Takashi Kikukawa, Naoki Kamo, Makoto Demura
    Optogenetics: Light-Sensing Proteins and their Applications 47 - 62 2015年01月01日 [査読有り][通常論文]
     
    Halorhodopsin is a light-driven inward Cl< sup> −< /sup> pump found in the membrane of a halophilic archaeon called Halobacterium salinarum. While the physiological role of halorhodopsin has not been fully resolved, its functional mechanism has been studied as a model system for anion transport. Halorhodopsin has become widely used in optogenetics due to its light-induced neural-silencing ability. Here, we summarize the functional analyses of halorhodopsin since its discovery. Like other microbial rhodopsins, halorhodopsin contains all-trans retinal bound to a specific lysine residue through a protonated Schiff base. Proton-pumping rhodopsins utilize Asp residues as the counter-ions for the protonated Schiff bases. In halorhodopsin, this Asp residue is replaced by Thr, and Cl< sup> −< /sup> becomes the counterion. Photoexcited halorhodopsin undergoes a photocycle including several intermediates where sequential Cl< sup> −< /sup> movements occur. During the formation of the N-intermediate, Cl< sup> −< /sup> moves from its original position to the cytoplasmic channel. During the subsequent N decay, the Cl< sup> −< /sup> is released to the cytoplasmic medium. During the Cl< sup> −< /sup> release, the dissociation constant of Cl< sup> −< /sup> increases significantly compared with that at the dark state. Next, another Cl< sup> −< /sup> is captured from the extracellular medium to complete the net Cl< sup> −< /sup> translocation. This recapture process is not well defined.
  • Munenori Furuse, Jun Tamogami, Toshiaki Hosaka, Takashi Kikukawa, Naoko Shinya, Masakatsu Hato, Noboru Ohsawa, So Young Kim, Kwang-Hwan Jung, Makoto Demura, Seiji Miyauchi, Naoki Kamo, Kazumi Shimono, Tomomi Kimura-Someya, Shigeyuki Yokoyama, Mikako Shirouzu
    Acta Crystallographica Section D: Biological Crystallography 71 Pt 11 2203 - 2216 2015年 [査読有り][通常論文]
     
    Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pK a of Glu206ARI, which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97ARI and Tyr221ARI on the cytoplasmic side, which facilitates the slow photocycle and regulates the pK a of Asp100ARI, a potential proton donor to the Schiff base, in the dark state.
  • Jun Tamogami, Katsunori Iwano, Atsushi Matsuyama, Takashi Kikukawa, Makoto Demura, Toshifumi Nara, Naoki Kamo
    JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 141 192 - 201 2014年12月 [査読有り][通常論文]
     
    Whether Cl- binds to the sensory rhodopsin II from Natronomonas pharaonis (NpSRII) that acts as a negative phototaxis receptor remains controversial. Two previous photoelectrochemical studies using SnO2 transparent electrodes and ATR-FTIR demonstrated that Cl- binding affects the photoinduced proton release from Asp193 in phospholipid (PC)-reconstituted NpSRII (Iwamoto et al., 2004; Kitade et al., 2009). In this study, we investigated the effects of Cl- on the photochemistry of NpSRII solubilized by detergent (DDM). Even under these conditions, Cl- could bind to NpSRII with a K-d of approximately 250 mM; this value is similar to 10-fold larger than that in the PC membrane. The binding of Cl- to NpSRII depended on the pH of the medium. In addition, Cl- binding induced the following effects: (1) a small red shift in the absorbance spectrum originating from the partial protonation of Asp75, (2) the formation of an interaction through a hydrogen-bonding network between Asp75 and Asp193, which is a proton-releasing residue, (3) several changes of the kinetic behavior of the photocycle, and (4) a photoinduced initial proton release from Asp193. The pK(a) values of Asp193 at various Cl- concentrations were also estimated. Based on the difference between the pKa values of Asp193 in Cl- bound and unbound NpSRII, the distance between the bound Cl- and Asp193 was determined to be approximately 6.1 angstrom, which agrees with the value estimated from the crystal structure presented by Royant et al. (2001). Therefore, the Cl- binding site affecting the photochemical properties of NpSRII is identical to the site proposed by Royant et al. (2001). This assignment was also supported by an experiment that introduced a mutation at Arg72. (C) 2014 Elsevier B.V. All rights reserved.
  • Takashi Tsukamoto, Makoto Demura, Yuki Sudo
    JOURNAL OF PHYSICAL CHEMISTRY B 118 43 12383 - 12394 2014年10月 [査読有り][通常論文]
     
    Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane alpha-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 degrees C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 degrees C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 degrees C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.
  • Kushibiki T, Kamiya M, Aizawa T, Kumaki Y, Kikukawa T, Mizuguchi M, Demura M, Kawabata S, Kawano K
    Biochimica et biophysica acta 1844 3 527 - 534 3 2014年03月 [査読有り][通常論文]
  • Takahiro Kushibiki, Masakatsu Kamiya, Tomoyasu Aizawa, Yasuhiro Kumaki, Takashi Kikukawa, Mineyuki Mizuguchi, Makoto Demura, Shun-Ichiro Kawabata, Keiichi Kawano
    Biochimica et Biophysica Acta - Proteins and Proteomics 1844 3 527 - 534 2014年03月 [査読有り][通常論文]
     
    Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS. © 2013 Elsevier B.V.
  • Kousuke Shibasaki, Hiroaki Shigemura, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOCHEMISTRY 52 51 9257 - 9268 2013年12月 [査読有り][通常論文]
     
    Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (P-i, i = 1-4) were adequate to describe the photocycle. The third component, P-3, was a quasi-equilibrium complex between the N and O intermediates, where a N <-> O + Cl- equilibrium was attained. The K-d,K-N <-> O values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, Delta V-N -> O, were estimated from the pressure dependence of K-d,K-N <-> O. A comparison between K-d,K-N <-> O and Delta V-N -> O led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K-d,K-N <-> O. For some mutants, however, large Delta V-N -> O values were found, whereas the K-d,K-N <-> O values were small. This suggests that the special coordination of a water molecule with the OH group of Thr is necessary for the increase in K-d,K-N <-> O. Mutants with a small K-d,K-N <-> O showed low pumping activities in the presence of inside negative membrane potential, while the mutant activities were not different in the absence of membrane potential. The effect of the mutation on the pumping activities is discussed.
  • Tetsuo Asakura, Yu Suzuki, Aya Nagano, David Knight, Masakatsu Kamiya, Makoto Demura
    BIOMACROMOLECULES 14 10 3731 - 3741 2013年10月 [査読有り][通常論文]
     
    The recombinant proteins [EE(A)(12)EETGRGDSPAAS](n) (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E-2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E-2(A)(n)E(2)TGRGDSPAE(2)(A)(n)E-2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO- to COOH of the E-2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)(12)EETGRGDSPAAS](n) (n = 5,10) by expression in Escherichia coli. The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E-2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)(12)EETGRGDSPAAS](10) has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material.
  • Takashi Tsukamoto, Xianglan Li, Hiromi Morita, Takashi Minowa, Tomoyasu Aizawa, Nobutaka Hanagata, Makoto Demura
    PLOS ONE 8 9 e75831  2013年09月 [査読有り][通常論文]
     
    Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.
  • Nakamura T, Aizawa T, Kariya R, Okada S, Demura M, Kawano K, Makabe K, Kuwajima K
    The Journal of biological chemistry 288 20 14408 - 14416 20 2013年05月 [査読有り][通常論文]
     
    Although HAMLET human alpha-lactalbumin made lethal to tumor cells), a complex formed by human alpha-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat alpha-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 degrees C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and beta(2)-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
  • Takashi Tsukamoto, Takashi Kikukawa, Takuro Kurata, Kwang-Hwan Jung, Naoki Kamo, Makoto Demura
    FEBS Letters 587 4 322 - 327 2013年02月14日 [査読有り][通常論文]
     
    Gloeobacter rhodopsin (GR) is a eubacterial proton pump having a highly conserved histidine near the retinal Schiff base counter-ion, aspartate. Various interactions between His and Asp of the eubacterial proton pump have been reported. Here, we showed the pH-dependent trimer/monomer transition of GR in the presence of dodecyl-β-d-maltoside by size-exclusion chromatography. The pH dependence was closely related to the protonation state of the counter-ion, Asp121. For the H87M mutant, pH dependence disappeared and a monomer became dominant. We concluded that the formation or breaking of the salt bridge between His87 and Asp121 inside the protein changes the quaternary structure. Structured summary of protein interactions: Rhodopsin and Rhodopsin bind by molecular sieving (View interaction) Rhodopsin and Rhodopsin bind by molecular sieving (View interaction: 1, 2) © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Saori Hayashi, Jun Tamogami, Takashi Kikukawa, Haruka Okamoto, Kazumi Shimono, Seiji Miyauchi, Makoto Demura, Toshifumi Nara, Naoki Kamo
    BIOPHYSICAL CHEMISTRY 172 61 - 67 2013年02月 [査読有り][通常論文]
     
    Halorhodopsin (HR), an inwardly directed, light-driven anion pump, is a membrane protein in halobacterial cells that contains the chromophore retinal, which binds to a specific lysine residue forming the Schiff base. An anion binds to the extracellular binding site near the Schiff base, and illumination makes this anion go to the intracellular channel, followed by its release from the protein and re-uptake from the opposite side. The thermodynamic properties of the anion binding in the dark, which have not been previously estimated, are determined using isothermal titration calorimetry (ITC). For Cl- as a typical substrate of HR from Natronomonas pharaonis, Delta G=-RT ln(1/K-d)=-15.9 kJ/mol, Delta H=-21.3 kJ/mol and T Delta S=-5.4 kJ/mol at 35 degrees C, where K-d represents the dissociation constant. In the dark, K-d values have been determined by the usual spectroscopic methods and are in agreement with the values estimated by ITC here. Opsin showed no Cl- binding ability, and the deprotonated Schiff base showed weak binding affinity, suggesting the importance of the positively charged protonated Schiff base for the anion binding. (C) 2013 Elsevier B.V. All rights reserved.
  • Satoshi Tomisawa, Chiharu Abe, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
    Biophysics (Japan) 9 79 - 83 2013年 [査読有り][通常論文]
     
    Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that pep-tide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method. © 2013 THE BIOPHYSICAL SOCIETY OF JAPAN.
  • Satoshi Tomisawa, Eri Hojo, Yoshitaka Umetsu, Shinya Ohki, Yusuke Kato, Mitsuhiro Miyazawa, Mineyuki Mizuguchi, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Tomoyasu Aizawa
    AMB EXPRESS 3 1 45  2013年 [査読有り][通常論文]
     
    Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.
  • Louisa Reissig, Tatsuya Iwata, Takashi Kikukawa, Makoto Demura, Naoki Kamo, Hideki Kandori, Yuki Sudo
    BIOCHEMISTRY 51 44 8802 - 8813 2012年11月 [査読有り][通常論文]
     
    In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color, tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol. 392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.
  • Jun Tamogami, Takashi Kikukawa, Toshifumi Nara, Kazumi Shimono, Makoto Demura, Naoki Kamo
    BIOCHEMISTRY 51 46 9290 - 9301 2012年11月 [査読有り][通常論文]
     
    Proteorhodopsin (PR) is one of the microbial rhodopsins that are found in marine eubacteria and likely functions as an outward light-driven proton pump. Previously, we [Tamogami, J., et al. (2009) Photochem. Photobiol. SS, 578-589] reported the occurrence of a photoinduced proton transfer in PR between pH 5 and 10 using a transparent ITO (indium-tin oxide) or SnO2 electrode that works as a time-resolving pH electrode. In the study presented here, the proton transfer at low pH (<4) was investigated. Under these conditions, Asp97, the primary counterion to the protonated Schiff base, is protonated. We observed a first proton release that was followed by an uptake; during this process, however, the M intermediate did not form. Through the use of experiments with several PR mutants, we found that Asp227 played an essential role in proton release. This residue corresponds to the Asp212 residue of bacteriorhodopsin, the so-called secondary Schiff base counterion. We estimated the pK(a) of this residue in both the dark and the proton-releasing photoproduct to be similar to 3.0 and similar to 2.3, respectively. The pK(a) value of Asp227 in the dark was also estimated spectroscopically and was approximately equal to that determined with the ITO experiments, which may imply the possibility of the release of a proton from Asp227. In the absence of Cl-, we observed the proton release in D227N and found that Asp97, the primary counterion, played a key role. It is inferred that the negative charge is required to stabilize the photoproducts through the deprotonation of Asp227 (first choice), the binding of Cl- (second choice), or the deprotonation of Asp97. The photoinduced proton release (possibly by the decrease in the pK(a) of the secondary counterion) in acidic media was also observed in other microbial rhodopsins with the exception of the Anabaena sensory rhodopsin, which lacks the dissociable residue at the position of Asp212 of BR or Asp227 of PR and halorhodopsin. The implication of this pK(a) decrease is discussed.
  • Yuji Furutani, Kuniyo Fujiwara, Tetsunari Kimura, Takashi Kikukawa, Makoto Demura, Hideki Kandori
    JOURNAL OF PHYSICAL CHEMISTRY LETTERS 3 20 2964 - 2969 2012年10月 [査読有り][通常論文]
     
    Ion transportation via the chloride ion pump protein pharaonis halorhodopsin (pHR) occurs through the sequential formation of several intermediates during a photocyclic reaction. Although the structural details of each intermediate state have been studied, the role of water molecules in the translocation of chloride ions inside of the protein at physiological temperatures remains unclear. To analyze the structural dynamics of water inside of the protein, we performed time-resolved Fourier transform infrared (FUR) spectroscopy under H2O or (H2O)-O-18 hydration and successfully assigned water O-H stretching bands. We found that a dangling water band at 3626 cm(-1) in pHR disappears in the L-1 and L-2 states. On the other hand, relatively intense positive bands at 3605 and 3608 cm(-1) emerged upon the formation of the X(N) and O states, respectively, suggesting that the chloride transportation is accompanied by dynamic rearrangement of the hydrogen-bonding network of the internal water molecules in pHR
  • Mineyuki Mizuguchi, Makoto Takeuchi, Shinya Ohki, Yuko Nabeshima, Takahide Kouno, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Katsuhide Yutani
    BIOCHEMISTRY 51 31 6089 - 6096 2012年08月 [査読有り][通常論文]
     
    The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 degrees C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement: data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the DI state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of alpha 4- and alpha 6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix helix association in the DI state. Therefore, in the folding process from the DI state to the native state, the alpha 4- and alpha 6-helices become separated and the central beta-sheet is folded between these helices. That is, the non-native interaction between the alpha 4- and a6-helices may be responsible for the unusually slow folding of PCP-0SH.
  • Kosuke Muroda, Keisuke Nakashima, Mikihiro Shibata, Makoto Demura, Hideki Kandori
    BIOCHEMISTRY 51 23 4677 - 4684 2012年06月 [査読有り][通常論文]
     
    Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.
  • Takashi Tsukamoto, Takanori Sasaki, Kazuhiro J. Fujimoto, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOPHYSICAL JOURNAL 102 12 2906 - 2915 2012年06月 [査読有り][通常論文]
     
    Halorhodopsin from NpHR is a light-driven Cl- pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl- release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl- transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl- in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl- transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Makoto Demura, Toshifumi Nara, Naoki Kamo
    JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 106 87 - 94 2012年01月 [査読有り][通常論文]
     
    Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration-time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370. (C) 2011 Elsevier B.V. All rights reserved.
  • Conststruction of a novel expression system for cryptdin-4 by using inclusion body formation.
    Sato Y, Tomisawa S, Aizawa T, Sakai N, Kamiya M, Kikukawa T, Kumaki Y, Demura M, Ayabe T, Kawano K
    Peptide Science 2011 393 - 394 2012年 [査読有り][通常論文]
  • Yamashita Y, Kikukawa T, Tsukamoto T, Kamiya M, Aizawa T, Kawano K, Miyauchi S, Kamo N, Demura M
    Biochimica et biophysica acta 1808 2905 - 2912 12 2011年12月 [査読有り][通常論文]
  • Higa, K, Takeshima, N, Moro, F, Kawakita, T, Kawashima, M, Demura, M, Shimazaki, J, Asakura, T, Tsubota, K, Shimmura, S
    J Biomater Sci 22 2261 - 2276 2011年11月19日 [査読無し][通常論文]
     
    Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin beta1 were expressed in the stratified epithelial sheets. Furt
  • Tetsuo Asakura, Hirohito Nishi, Aya Nagano, Ai Yoshida, Yasumoto Nakazawa, Masakatsu Kamiya, Makoto Demura
    BIOMACROMOLECULES 12 11 3910 - 3916 2011年11月 [査読有り][通常論文]
     
    It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF10, consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The H-1, N-15, and C-13 chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a C-13 solid-state NMR study was performed on a C-13 selectively labeled model peptide, AGSGAG[3-C-13]A(7)GSGAGAGSGGT[2-C-13]G(19)R[1-C-13]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the beta-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins.
  • Gang Dai, Yu Zhang, Jun Tamogami, Makoto Demura, Naoki Kamo, Hideki Kandori, Tatsuo Iwasa
    BIOCHEMISTRY 50 33 7177 - 7183 2011年08月 [査読有り][通常論文]
     
    Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (lambda(max)) at 482 nm and can thermally revert back to spR above -160 degrees C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9=C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.
  • Sasaki T, Demura M, Kato N, Mukai Y
    Biochemistry 50 12 2283 - 2290 12 2011年03月29日 [査読有り][通常論文]
     
    A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-d-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR. © 2011 American Chemical Society.
  • Kazunari Higa, Naomi Takeshima, Fumika Moro, Tetsuya Kawakita, Motoko Kawashima, Makoto Demura, Jun Shimazaki, Tetsuo Asakura, Kazuo Tsubota, Shigeto Shimmura
    JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 22 17 2261 - 2276 2011年 [査読有り][通常論文]
     
    Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin beta 1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium. (C) Koninklijke Brill NV, Leiden, 2011
  • Takahide Kouno, Nobuhisa Watanabe, Naoki Sakai, Takashi Nakamura, Yuko Nabeshima, Masashi Morita, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Tsuneo Imanaka, Isao Tanaka, Keiichi Kawano
    JOURNAL OF MOLECULAR BIOLOGY 405 2 560 - 569 2011年01月 [査読有り][通常論文]
     
    Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a beta-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the beta-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down beta-sheets. This up-and-down beta-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down beta-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down beta-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain. (C) 2010 Elsevier Ltd. All rights reserved.
  • Wei Wang, Soichiro Itoh, Tomoyasu Aizawa, Atsushi Okawa, Katsuyoshi Sakai, Tsuneo Ohkuma, Makoto Demura
    BIOMEDICAL MATERIALS 5 6 065009  2010年12月 [査読有り][通常論文]
     
    A chitosan/marine-originated collagen composite has been developed. This composite gel was characterized and its biocompatibility, as well as an inflammatory reaction, was observed. The chitosan gel including N-3-carboxypropanoil-6-O-(carboxymethyl) chitosan of 3 mol%, 6-O-(carboxymethyl) chitosan of 62 mol% and 6-O-(carboxymethyl) chitin of 35 mol% was prepared and compounded with the salmon atelocollagen (SA) gel at different mixture ratios. The composite gels were injected subcutaneously in to the back of rats. The specimens were harvested for a histological survey as well as a tumor necrosis factor-alpha (TNF-alpha) assay by ELISA. The inflammatory cell infiltration and release of TNF-alpha were successively controlled low with the ratio of SA to chitosan at 10:90 or 20:80. The SA gel first, within 2 weeks, and then chitosan in the composite gel were slowly absorbed after implantation, followed by soft tissue formation. It is expected that this composite gel will be available as a carrier for tissue filler and drug delivery systems.
  • Yasuhiro Nonaka, Hideki Muto, Tomoyasu Aizawa, Etsuro Okabe, Shohei Myoba, Takuya Yokoyama, Shin Saito, Fumie Tatami, Yasuhiro Kumaki, Masakatsu Kamiya, Takashi Kikukawa, Mineyuki Mizuguchi, Shigeharu Takiya, Masataka Kinjo, Makoto Demura, Keiichi Kawano
    BIOCHEMISTRY 49 38 8367 - 8375 2010年09月 [査読有り][通常論文]
     
    The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and a-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in a-helix structure content.
  • Takahashi M, Mizuguchi M, Shinoda H, Aizawa T, Demura M, Okazawa H, Kawano K
    Biochimica et biophysica acta 1804 1500 - 1507 7 2010年07月 [査読有り][通常論文]
  • Masakatsu Kamiya, Keisuke Oyauchi, Yoshinori Sato, Takuya Yokoyama, Mofei Wang, Tomoyasu Aizawa, Yasuhiro Kumaki, Mineyuki Mizuguchi, Kunio Imai, Makoto Demura, Koichi Suzuki, Keiichi Kawano
    JOURNAL OF PEPTIDE SCIENCE 16 5 242 - 248 2010年05月 [査読有り][通常論文]
     
    We previously reported that yamamarin, a pentapeptide with an amidated C-terminus (DILRG-NH(2)) isolated from larvae of the silkmoth, and its palmitoylated analog (C16-DILRG-NH(2)) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure-activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C-terminal part (-RG-NH(2)) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG-NH(2) and C16-DILRG-NH(2) revealed that the N-terminal palmitoyl group of C16-DILRG-NH(2) did not affect the conformation of the C-terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright (C) 2010 European Peptide Society and John Wiley & Sons, Ltd.
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Ayaka Takemura, Makoto Demura, Naoki Kamo
    BIOPHYSICAL JOURNAL 98 7 1353 - 1363 2010年04月 [査読有り][通常論文]
     
    Sensory rhodopsin II (HsSRII, also called phoborhodopsin) is a negative phototaxis receptor of Halobacterium salinarum, a bacterium that avoids blue-green light. In this study, we expressed the protein in Escherichia colt cells, and reconstituted the purified protein with phosphatidylcholine. The reconstituted HsSRII was stable. We examined the photocycle by flash-photolysis spectroscopy in the time range of milliseconds to seconds, and measured proton uptake/release using a transparent indium-tin oxide electrode. The pKa of the counterion of the Schiff base, Asp(73), was 3.0. Below pH 3, the depleted band was observed on flash illumination, but the positive band in the difference spectra was not found. Above pH 3, the basic photocycle was HsSRII (490) -> M (350) -> O (520) -> Y (490) -> HsSRII, where the numbers in parentheses are the maximum wavelengths. The decay rate of O-intermediate and Y-intermediate were pH-independent, whereas the M-intermediate decay was pH-dependent. For 3 < pH < 4.5, the M-decay was one phase, and the rate decreased with an increase in pH. For 4.5 < pH < 6.5, the decay was one phase with pH-independent rates, and azide markedly accelerated the M-decay. These findings suggest the existence of a protonated amino acid residue (X-H) that may serve as a proton relay to reprotonate the Schiff base. Above pH 6.5, the M-decay showed two phases. The fast M-decay was pH-independent and originated from the molecule having a protonated X-H, and the slow M-decay originated from the molecule having a deprotonated X, in which the proton came directly from the outside. The analysis yielded a value of 7.5 for the pKa of X-H. The proton uptake and release occurred during M-decay and O-decay, respectively.
  • Takanori Sasaki, Tomoyasu Aizawa, Masakatsu Kamiya, Takashi Kikukawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOCHEMISTRY 48 51 12089 - 12095 2009年12月 [査読有り][通常論文]
     
    Halorhodopsin from Natronomonas pharaonis (NpHR) acts an inward-directed, light-driven chloride pump and forms a homotrimer. To evaluate effect of trimeric assembly, that is, intermolecular interaction, on the control or modulation of light-driven chloride pumping activity of individual HRs, it is important to understand the thermal and chloride sensitivity of trimer dissociation and the structural stability of HR. In this study, the thermal dissociation of NpHR trimer to monomer in a dodecyl beta-D-maltoside-solubilized system was investigated, using size-exclusion chromatography and visible absorption. In the absence of Cl(-), NpHR retained the trimer assembly at 25 degrees C but dissociated to the monomer with an increase in temperature to > 40 degrees C. Oil the other hand, in the presence of Cl-, the trimer assembly was maintained at 40 degrees C. The dissociation of the trimer to the monomer after incubation at 40 degrees C, which wits determined via size-exclusion chromatography, depended oil the Cl- concentration and showed a sigmoidal isotherm. From this isotherm, the apparent dissociation constant for Cl(-) was estimated to be 22 mM with a Hill coefficient of 2.2. A similar isotherm was obtained when SO(4)(2-) was used instead of Cl- with a dissociation constant of 94 mM. On the other hand, thermal dissociation of the NpHR trimer to the monomer in the absence of Cl(-) proceeded by two components: the fast component is Susceptible to the changes in temperature and detergent concentration, and the slow component is accompanied by bleaching at the same time. Activation energies of the fast and slow dissociation components and bleaching were 57.8, 35.3, and 40.5 kcal/mol, respectively. The presence of a second chloride-binding site with a Hill coefficient of similar to 2 at file surface of NpHR to control the trimer-monomer conversion was discussed.
  • Yoshitaka Umetsu, Tomoyasu Aizawa, Kaori Muto, Hiroko Yamamoto, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 43 29625 - 29634 2009年10月 [査読有り][通常論文]
     
    Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.
  • Takahide Kouno, Mineyuki Mizuguchi, Tomoyasu Aizawa, Hiroyuki Shinoda, Makoto Demura, Shun-ichiro Kawabata, Keiichi Kawano
    BIOCHEMISTRY 48 32 7629 - 7635 2009年08月 [査読有り][通常論文]
     
    Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. The amino acid sequence of big defensin is divided into an N-terminal hydrophobic domain and a C-terminal cationic domain, Which are responsible for antimicrobial activities against Gram-positive and -negative bacteria, respectively. The N-terminal domain of big defensin forms a unique globular conformation with two alpha-helices and a parallel beta-sheet, while the C-terminal domain adopts a beta-defensin-like fold. Although Our previous study implied that big defensin changes its N-terminal structure in a micellar environment, due to the poor quality of the NMR spectra it remained to be resolved whether the N-terminal domain adopts any Structure in the presence of micelles. In this analysis, We Successfully determined the structure of the N-terminal fragment of big defensin in a micellar solution, showing that the fragment peptide forms a single a-helix structure. Moreover, NMR experiments using paramagnetic probes revealed that the N-terminal domain of big defensin penetrates into the micelle with a dipping at the N-terminal edge of the alpha-helix. Here, we propose a model for how big defensin associates with the target membrane.
  • Shigeharu Takiya, Shin Saito, Takuya Yokoyama, Daisuke Matsumoto, Tomoyasu Aizawa, Masakatsu Kamiya, Makoto Demura, Keiichi Kawano
    JOURNAL OF BIOCHEMISTRY 146 1 103 - 111 2009年07月 [査読有り][通常論文]
     
    The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino-acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.
  • Takahashi M, Mizuguchi M, Shinoda H, Aizawa T, Demura M, Okazawa H, Kawano K
    Biochimica et biophysica acta 1794 936 - 943 6 2009年06月 [査読有り][通常論文]
  • Keisuke Nakashima, Takumi Nakamura, Satoshi Takeuchi, Mikihiro Shibata, Makoto Demura, Tahei Tahara, Hideki Kandori
    JOURNAL OF PHYSICAL CHEMISTRY B 113 24 8429 - 8434 2009年06月 [査読有り][通常論文]
     
    Halorhodopsin (HR) is a light-driven chloride pump. Cl- is bound in the Schiff base region of the retinal chromophore, and unidirectional Cl- transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. It is known that HR from Natronobacterium pharaonis (pHR) also pumps NO3- with similar efficiency, suggesting that NO3- binds to the Cl--binding site. In the present study, we investigated the properties of the anion-binding site by means of ultrafast pump-probe spectroscopy and low-temperature FTIR spectroscopy. The obtained data were surprisingly similar between pHR-NO3- and pHR-Cl-, even though the shapes and sizes of the two anions are quite different. Femtosecond pump-probe spectroscopy showed very similar excited-state dynamics between pHR-NO3- and pHR-Cl-. Low-temperature FTIR spectroscopy of unlabeled and [zeta-N-15]Lys-labeled pHR revealed almost identical hydrogen-bonding strengths of the protonated retinal Schiff base between pHR-NO3- and pHR-Cl-, which is similarly strengthened after retinal isomerization. There were spectral variations for water stretching vibrations between pHR-NO3- and pHR-Cl-, suggesting that the water molecules hydrate each anion. Nevertheless, the overall spectral features were similar for the two species. These observations strongly suggest that the anion-binding site has a flexible structure and that the interaction between retinal and the anions is weak, despite the presence of an electrostatic interaction. Such a flexible hydrogen-bonding network in the Schiff base region in HR appears to be in remarkable contrast to that in light-driven proton-pumping proteins.
  • Shin-ichi Nakatogawa, Yasunori Oda, Masakatsu Kamiya, Tatsuro Kamijima, Tomoyasu Aizawa, Kevin D. Clark, Makoto Demura, Keiichi Kawano, Michael R. Strand, Yoichi Hayakawa
    CURRENT BIOLOGY 19 9 779 - 785 2009年05月 [査読有り][通常論文]
     
    Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.
  • Keiichi Inoue, Megumi Kubo, Makoto Demura, Naoki Kamo, Masahide Terazima
    BIOPHYSICAL JOURNAL 96 9 3724 - 3734 2009年05月 [査読有り][通常論文]
     
    Reaction dynamics of a chloride ion pump protein, halorhodopsin (HR), from Natronomonas pharaonis (N. pharaonis) (NpHR) was studied by the pulsed-laser-induced transient grating (TG) method. A detailed investigation of the TG signal revealed that there is a spectrally silent diffusion process besides the absorption-observable reaction dynamics. We interpreted these dynamics in terms of release, diffusion, and uptake of the Cl- ion. From a quantitative global analysis of the signals at various grating wavenumbers, it was concluded that the release of the Cl- ion is associated with the L2 -> (L2 (or N) reversible arrow O) process, and uptake of Cl- occurs with the (L2 (or N) reversible arrow O) -> NpHR' process. The diffusion coefficient of NpHR solubilized in a detergent did not change during the cyclic reaction. This result contrasts the behavior of many photosensor proteins and implies that the change in the H-bond network from intra- to intermolecular is not significant for the activity of this protein pump.
  • Yasuhiro Nonaka, Daisuke Akieda, Tomoyasu Aizawa, Nobuhisa Watanabe, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Takashi Kikukawa, Makoto Demura, Keiichi Kawano
    FEBS JOURNAL 276 8 2192 - 2200 2009年04月 [査読有り][通常論文]
     
    In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.
  • Megumi Kubo, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 2 547 - 555 2009年03月 [査読有り][通常論文]
     
    Halorhodopsin (HR) acts as a light-driven chloride pump which transports a chloride ion from the extracellular (EC) to the cytoplasmic space during a photocycle reaction that includes some photointermediates initiated by illumination. To understand the chloride uptake mechanisms, we focused on a basic residue Arg123 of HR from Natronomonas pharaonis (NpHR), which is the only basic residue located in the EC half ion channel. By the measurements of the visible absorption spectra in the dark and the light-induced inward current through the membrane, it was shown that the chloride binding and transport ability of NpHR completely disappeared by the change of arginine to glutamine. From flashphotolysis analysis, the photocycle of R123Q differed from that of wildtype NpHR completely. The response of the R123H mutant depended on pH. These facts imply that the positive charge at position 123 is essential for chloride binding in the ground state and for the chloride uptake under illumination. On the basis of the molecular structures of HR and the anion-transportable mutants of bacteriorhodopsin, the effects of the positive charge and the conformational change of the Arg123 side chain as well as the chloride-pumping mechanism are discussed.
  • Yasuhiro Nonaka, Daisuke Akieda, Nobuhisa Watanabe, Masakatsu Kamiya, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano
    Biophysical Journal 96 3 67a - 68a 2009年02月
  • Takanori Sasaki, Megumi Kubo, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 1 130 - 136 2009年01月 [査読有り][通常論文]
     
    Halorhodopsin (HR) is a transmembrane seven-helix retinal protein, and acts as an inward light-driven Cl(-) pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 mm) to 1% (20 mm) dodecyl-beta-d-maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 mm). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton-coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR-DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross-linked NpHR by glutaraldehyde gave the SDS-PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR (Escherichia coli and Halobacterium salinarum), CD spectra showed exciton-coupling, which suggests strongly the trimer structure in the cell membrane.
  • Tatsuro Kamijima, Ayaka Ohmura, Toshiya Sato, Kaoru Akimoto, Miki Itabashi, Mineyuki Mizuguchi, Masakatsu Kamiya, Takashi Kikukawa, Tomoyasu Aizawa, Masayuki Takahashi, Keiichi Kawano, Makoto Demura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 376 1 211 - 214 2008年11月 [査読有り][通常論文]
     
    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, Circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes. (C) 2008 Elsevier Inc. All rights reserved.
  • Takahide Kouno, Mineyuki Mizuguchi, Masakiyo Sakaguchi, Eiichi Makino, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Nam-Ho Huh, Keiichi Kawano
    JOURNAL OF PEPTIDE SCIENCE 14 10 1129 - 1138 2008年10月 [査読有り][通常論文]
     
    S100A11 protein is a member of the S100 family containing two EF-hand motifs. It undergoes phophorylation on residue T10 after cell stimulation such as an increase in Ca2+ concentration. Phosphorylated S100A11 I can be recognized by its target protein, nucleolin. Although S100A11 is initially expressed in the cytoplasm, it is transported to the nucleus by the action of nucleolin. In the nucleus, S100A11 suppresses the growth of keratinocytes through p21(CIP1/WAF1) activation and induces cell differentiation. Interestingly, the N-terminal fragment of S100A11 has the same activity as the full-length protein: i.e. it is phosphorylated in vivo and binds to nucleolin. In addition, this fragment leads to the arrest of cultured keratinocyte growth. We examined the solution structure of this fragment peptide and explored its structural properties before and after phosphorylation. In a trifluoroethanol solution, the peptide adopts the alpha-helical structure just as the corresponding region of the full-length S100A11. Phosphorylation induces a disruption of the N-capping conformation of the alpha-helix, and has a tendency to perturb its surrounding structure. Therefore, the phosphorylated threonine lies in the N-terminal edge of the alpha-helix. This local structural change can reasonably explain why the phosphorylation of a residue that is initially buried in the interior of protein allows it to be recognized by the binding partner. Copyright (C) 2008 European Peptide Society and John Wiley & Sons, Ltd.
  • Takahide Kouno, Naoki Fujitani, Mineyuki Mizuguchi, Tsukasa Osaki, Shin-ichiro Nishimura, Shun-ichiro Kawabata, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta, Keiichi Kawan
    BIOCHEMISTRY 47 40 10611 - 10619 2008年10月 [査読有り][通常論文]
     
    Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a P-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.
  • Takumi Nakamura, Satoshi Takeuchi, Mikihiro Shibata, Makoto Demura, Hideki Kandori, Tahei Tahara
    JOURNAL OF PHYSICAL CHEMISTRY B 112 40 12795 - 12800 2008年10月 [査読有り][通常論文]
     
    Halorhodopsin is a retinal protein that acts as a light-driven chloride pump in the Haloarchaeal cell membrane. A chloride ion is bound near the retinal chromophore, and light-induced all-trans -> 13-cis isomerization triggers the unidirectional chloride ion pump. We investigated the primary ultrafast dynamics of Natronomonas pharaonis halorhodopsin that contains Cl-, Br-, or I- (pHR-Cl-, pHR-Br-, or pHR-I-) using ultrafast pump-probe spectroscopy with similar to 30 fs time resolution. All of the temporal behaviors of the S-n <- S, absorption, ground-state bleaching, K intermediate (13-cis form) absorption, and stimulated emission were observed. In agreement with previous reports, the primary process exhibited three dynamics. The first dynamics corresponds to the population branching process from the Franck-Condon (FC) region to the reactive (S-1(r)) and non-reactive (S-1(nr)) S, states. With the improved time resolution, it was revealed that the time constant of this branching process (tau(1)) is as short as 50 fs. The second dynamics was the isomerization process of the S-1(r) state to generate the ground-state 13-cis form, and the time constant (tau(2)) exhibited significant halide ion dependence (1.4, 1.6, and 2.2 ps for pHR-Cl-, pHR-Br-, and pHR-I-, respectively). The relative quantum yield of the isomerization, which was evaluated from the pump-probe signal after 20 ps, also showed halide ion dependence (1.00, 1. 14, and 1.35 for pHR-Cl-, pHR-Br-, and pHR-I-, respectively). It was revealed that the halide ion that accelerates isomerization dynamics provides the lower isomerization yield. This finding suggests that there is an activation barrier along the isomerization coordinate on the S, potential energy surface, meaning that the three-state model, which is now accepted for bacteriorhodopsin, is more relevant than the two-state model for the isomerization process of halorhodopsin. We concluded that, with the three-state model, the isomerization rate is controlled by the height of the activation barrier on the S, potential energy surface while the overall isomerization yield is determined by the branching ratios at the FC region and the conical intersection. The third dynamics attributable to the internal conversion of the S-1(nr) state also showed notable halide ion dependence (tau(3) = 4.5, 4.6, and 6.3 ps for pHR-Cl-, pHR-Br-, and pHR-I-). This suggests that some geometrical change may be involved in the relaxation process of the S-1(nr) state.
  • Shin Saito, Takuya Yokoyama, Tomoyasu Aizawa, Kyosuke Kawaguchi, Takeshi Yamaki, Daisuke Matsumoto, Tatsuro Kamijima, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Sigeharu Takiya, Makoto Demura, Keiichi Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 1 414 - 426 2008年07月 [査読有り][通常論文]
     
    Fibroin-modulator-binding protein 1 (FMBP-1) is a predicted transcription factor of the silkworm fibroin gene. The DNA-binding domain of FMBP-1 consists of four almost perfect tandem repeats of 23 amino acids each (R1-R4), and is referred to as the score and three amino acid peptide repeat (STPR) domain. This characteristic domain is conserved in eukaryotes, but the DNA-binding mode is not known. In this study, the structural properties of the DNA-bound form of the STPR domain were characterized. The combined experiments indicated that the STPR domain bound to the DNA duplex wit a 1:1 binding ratio. The specific DNA caused considerable changes in the thermal unfolding profile and the digestion pattern of the STPR domain. These data suggested that the domain adapts a quite rigid helix-rich structure in the DNA-bound state, even though it moves flexibly in the absence of DNA. Furthermore, mutual induced-fit conformational change was also observed in DNA. Finally, we determined the DNA-binding surface of the STPR third repeat (R3) by alamne scanning mutagenesis; a particular site, composed of hydrophobic and hydrophilic residues, was identified. Notably, the substitution of Arg-9 in R3 with alanine residue, which is located in the middle of the surface, drastically abolished the -i-helix-inducing and DNA-binding abilities. From these results, we predicted the DNA-binding mode of the STPR domain.
  • Yasuhiro Nonaka, Tomoyasu Aizawa, Daisuke Akieda, Masanori Yasui, Masahiro Watanabe, Nobuhisa Watanabe, Isao Tanaka, Masakatsu Kamiya, Mineyuki Mizuguchi, Makoto Demura, Keiichi Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 1 313 - 322 2008年07月 [査読有り][通常論文]
     
    Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.
  • Wei Wang, Soichiro Itoh, Atsushi Matsuda, Tomoyasu Aizawa, Makoto Demura, Shizuko Ichinose, Kenichi Shinomiya, Junzo Tanaka
    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 85A 4 919 - 928 2008年06月 [査読有り][通常論文]
     
    We have developed a novel bilayered chitosan tube that comprises an outer layer of chitosan film and an inner layer of chitosan nonwoven nano/microfiber mesh. The tube is fabricated with an electrospinning method. We characterized the microstructure and mechanical properties of this material. We introduced glycine spacers into the CYIGSR sequence, domain of laminin-1 that enhances Schwann cells migration and attachment, as well as neural outgrowth, resulting in the amino acid sequences CGGYIGSR and CGGGGGGYIGSR. These peptides were covalently bound to the nano/microfiber mesh surface of the chitosan tube to examine the effects of peptide mobility on nerve regeneration. Scaffolds constructed from these bilayered chitosan tubes were grafted to bridge injured sciatic nerve. Isografting was performed as a control. These scaffolds were removed 5 and 10 weeks after implantation for histological analysis. Nerve regeneration into chitosan tubes, on which the CGGGGGGYIGSR peptide was immobilized, exhibited efficacy similar to that of the isograft and represent a promising candidate for promoting peripheral nerve repair. (C) 2007 Wiley Periodicals, Inc.
  • D. Akieda, Y. Nonaka, N. Watanabe, I. Tanaka, M. Kamiya, T. Aizawa, K. Nitta, M. Demura, K. Kawano
    2008年05月27日
  • Seiji Miyauchi, Akiteru Seki, Kentaro Aoyama, Takashi Kikukawa, Makoto Demura, Vadivel Ganapathy, Naoki Kamo
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 133 - 134 2008年 [査読有り][通常論文]
  • Takahide Kouno, Mineyuki Mizuguchi, Hiromasa Tanaka, Ping Yang, Yoshihiro Mori, Hiroyuki Shinoda, Kana Unoki, Tomoyasu Aizawa, Makoto Demura, Koichi Suzuki, Keiichi Kawano
    BIOCHEMISTRY 46 48 13733 - 13741 2007年12月 [査読有り][通常論文]
     
    Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ Channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional H-1 nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungai peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.
  • Y. Nonaka, D. Akieda, N. Watanabe, I. Tanaka, M. Kamiya, T. Aizawa, K. Nitta, M. Demura, K. Kawano
    2007年11月27日
  • Hiroyasu Nakatani, Kosuke Maki, Kimiko Saeki, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Shuji Tomoda, Kunihiro Kuwajima
    BIOCHEMISTRY 46 17 5238 - 5251 2007年05月 [査読有り][通常論文]
     
    The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The Phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.
  • Naoki Fujitani, Takahide Kouno, Taku Nakahara, Kenji Takaya, Tsukasa Osaki, Shun-Ichiro Kawabata, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Shin-Ichiro Nishimura, Keiichi Kawano
    JOURNAL OF PEPTIDE SCIENCE 13 4 269 - 279 2007年04月 [査読有り][通常論文]
     
    Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 angstrom, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.
  • Chisa Hasegawa, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Yuki Sudo, Megumi Kubo, Makoto Demura, Naoki Kamo
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 83 2 293 - 302 2007年03月 [査読有り][通常論文]
     
    An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [20061 1274). Previously, we reported that the phR double mutant, P240T/F250Y(phR), can bind with pHtrII. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR -> P-1 -> P2 -> P3 -> P-4 -> phR. The P-3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P-3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation because of the inhibition of formation of the X-intermediate.
  • Makoto Takeuchi, Mineyuki Mizuguchi, Takahide Kouno, Yoshinori Shinohara, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Keiichi Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 66 3 716 - 725 2007年02月 [査読有り][通常論文]
     
    Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin. Proteins 2007;66:716-725. (c) 2006 Wiley-Liss, Inc.
  • Seiji Miyauchi, Akiteru Seki, Saori Hayashi, Takashi Kikukawa, Maki Satoh, Makoto Demura, Vadivel Ganapathy, Naoki Kamo
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 127 163 - 164 2007年 [査読有り][通常論文]
  • Mikihiro Shibata, Yuko Saito, Makoto Demura, Hideki Kandori
    Chemical Physics Letters 432 4-6 545 - 547 2006年12月
  • Masanori Yasui, Taku Miyahara, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    PROTEIN JOURNAL 25 7-8 475 - 482 2006年12月 [査読有り][通常論文]
     
    In order to investigate the thermodynamics of the unfolding of metalloproteins, the thermal denaturation of bovine alpha-lactalbumin (BLA), a typical calcium-binding protein, was investigated under a wide variety of calcium ion activities by means of differential scanning calorimetry. The excess heat capacity obtained as above is composed of those of the following three reactions: (i) the release of a calcium ion from holo-BLA; (ii) the capture of the released calcium ion by the chelating reagent; and (iii) the denaturation of native apo-BLA. The results indicated that the presence of the chelating reagent had a remarkable effect on the apparent enthalpy change for the denaturation of holo-BLA. On the other hand, the influence of the chelator on the heat capacity change was shown to be negligible. Because the denaturation reaction of holo-BLA includes Reactions (i) and (iii), it had to be handled as a three-state reaction. Such an investigation of the unfolding has been scarcely found that the activity of the metal ion is controlled precisely in wide range.
  • Masanori Yasui, Taku Miyahara, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    The Protein Journal 25 7-8 475 - 482 2006年11月28日
  • Ken Arita, Masashi Akiyama, Tomoyasu Aizawa, Yoshitaka Umetsu, Ikuo Segawa, Maki Goto, Daisuke Sawamura, Makoto Demura, Keiichi Kawano, Hiroshi Shimizu
    AMERICAN JOURNAL OF PATHOLOGY 169 2 416 - 423 2006年08月 [査読有り][通常論文]
     
    Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient's keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional H-1 nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication.
  • Norikazu Muneda, Mikihiro Shibata, Makoto Demura, Hideki Kandori
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 128 19 6294 - 6295 2006年05月 [査読有り][通常論文]
  • Satoshi Watanabe, Masahito Tada, Tomoyasu Aizawa, Masanobu Yoshida, Tadamasa Sugaya, Makoto Taguchi, Takahide Kouno, Takashi Nakamura, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano
    PROTEIN AND PEPTIDE LETTERS 13 8 815 - 822 2006年 [査読有り][通常論文]
     
    GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The H-1 NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core P-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn(2). Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.
  • Masahito Tada, Yoshinori Shinohara, Ichiro Kato, Koichi Hiraga, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Mineyuki Mizuguchi, Keiichi Kawano
    ACTA HISTOCHEMICA ET CYTOCHEMICA 39 2 31 - 34 2006年 [査読有り][通常論文]
     
    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein ( GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.
  • M Mizuguchi, A Matsuura, Y Nabeshima, K Masaki, M Watanabe, T Aizawa, M Demura, K Nitta, Y Mori, H Shinoda, K Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 61 2 356 - 365 2005年11月 [査読有り][通常論文]
     
    The N-terminal half of the a-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of a-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.
  • M Kubo, M Sato, T Aizawa, C Kojima, N Kamo, M Mizuguchi, K Kawano, M Demura
    BIOCHEMISTRY 44 39 12923 - 12931 2005年10月 [査読有り][通常論文]
     
    Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-D-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-D-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 MM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <-> OG-NpHR (disassembled) <-> intermediate -> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.
  • T Sasaki, M Sonoyama, M Demura, S Mitaku
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 81 5 1131 - 1137 2005年09月 [査読有り][通常論文]
     
    In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.
  • M Shibata, N Muneda, T Sasaki, K Shimono, N Kamo, M Demura, H Kandori
    BIOCHEMISTRY 44 37 12279 - 12286 2005年09月 [査読有り][通常論文]
     
    Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D2O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl- only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L, intermediate, whereas the halide dependence revealed that the acceptor is not Cl-, but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl- is not a driving force of the motion of Cl-. Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl- less polar in the L-1 intermediate, which presumably drives the motion of Cl- from its binding site to the cytoplasmic domain.
  • M Demura, T Yoshida, T Hirokawa, Y Kumaki, T Aizawa, K Nitta, Bitter, I, K Toth
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 15 5 1367 - 1370 2005年03月 [査読有り][通常論文]
     
    The molecular recognition of neurotransmitters, dopamine and acetylcholine with an amphiphilic resorcinarene receptor was investigated in an aqueous sodium dodecylsulfate (SDS) micelle system by H-1 NMR measurements. The interaction distances of these neurotransmitters from the hydrophilic cavity of the amphiphilic receptor were estimated based on the calculation of the ring current shift using the atomic coordinates obtained from molecular dynamics calculation. (c) 2005 Elsevier Ltd. All rights reserved.
  • M Sato, M Kubo, T Aizawa, N Kamo, T Kikukawa, K Nitta, M Demura
    BIOCHEMISTRY 44 12 4775 - 4784 2005年03月 [査読有り][通常論文]
     
    Natronomonas (Natronobacterium.) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl- ion pump. For efficient Cl- transport, the existence of Cl-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl- to anion-free NpHR. The binding affinity was affected in Mutants in which certain EC residues had been replaced; this finding revealed the importance of Ar123. On the other hand, Mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC Mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants. L2 -photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl- to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.
  • Masanobu Yoshida, Kunio Shitara, Kimiaki Matsubara, Takahide Kouno, Tomoyasu Aizawa, Yoichi Hayakawa, Yasuhiro Kuniaki, Mineyuki Mizuguchi, Makoto Demura, Katsutoshi Nitta, Keiichi Kawano
    Peptides 2004, Proceedings 738 - 739 2005年 [査読無し][通常論文]
  • M Yoshida, T Aizawa, T Nakamura, K Shitara, Y Hayakawa, K Matsubara, K Miura, T Kouno, KD Clark, MR Strand, M Mizuguchi, M Demura, K Nitta, K Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 49 51331 - 51337 2004年12月 [査読有り][通常論文]
     
    Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe(3)) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly(5) or Gly(6) in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe(3) is a binding determinant of the N-terminal domain.
  • Watanabe M, Aizawa T, Demura M, Nitta K
    Biochimica et biophysica acta 1702 2 129 - 136 2 2004年11月 [査読有り][通常論文]
     
    The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degrees C. The DeltaV values at 25 degrees C were obtained for the N-MG (-46 cm3/mol) and MG-unfolded-state (U) transition (-40 cm3/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm3/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate.
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta
    PROTEIN AND PEPTIDE LETTERS 11 4 325 - 330 2004年08月 [査読有り][通常論文]
     
    The effect of pressure on the unfolding of the molten globule (MG) state of canine milk lysozyme (CML) was examined Using ultraviolet spectroscopy. The volume changes of the MG-unfolded-state transition were observed at pH 2.0 and around 20 to 60degreesC. but 110 volume change has been found for bovine alpha-lactalbumin, Which is homologous to CML. Our results suggest that the MG state of CML possesses a tightly packed hydrophobic core.
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta
    PROTEIN JOURNAL 23 5 335 - 342 2004年07月 [査読有り][通常論文]
     
    The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.
  • M Sato, T Kikukawa, T Araiso, H Okita, K Shimono, N Kamo, M Demura, K Nitta
    JOURNAL OF BIOCHEMISTRY 134 1 151 - 158 2003年07月 [査読有り][通常論文]
     
    Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl-binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl- ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.
  • M Sato, T Kikukawa, T Araiso, H Okita, K Shimono, N Kamo, M Demura, K Nitta
    BIOPHYSICAL CHEMISTRY 104 1 209 - 216 2003年05月 [査読有り][通常論文]
     
    Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift Of A a upon Cl- addition, from which the dissociation constants of Cl- were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degreesC. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorbodopsin is important for the chloride binding. (C) 2003 Elsevier Science B.V. All rights reserved.
  • N Fujitani, M Kanagawa, T Aizawa, T Ohkubo, S Kaya, M Demura, K Kawano, S Nishimura, K Taniguchi, K Nitta
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 1 223 - 229 2003年01月 [査読有り][通常論文]
     
    It has been well established that phosphotylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase. reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H+/K+-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation. and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H+/K+-ATPase. (C) 2002 Elsevier Science (USA). All rights reserved.
  • K Sasahara, M Demura, K Nitta
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 49 4 472 - 482 2002年12月 [査読有り][通常論文]
     
    The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25degreesC under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (< 10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<---->I<---->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed. (C) 2002 Wiley-Liss, Inc.
  • M Tada, Y Kobashigawa, M Mizuguchi, K Miura, T Kouno, Y Kumaki, M Demura, K Nitta, K Kawano
    BIOCHEMISTRY 41 46 13807 - 13813 2002年11月 [査読有り][通常論文]
     
    Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine (x-lactalbumin. We used model-free analysis based on N-15 nuclear magnetic resonance relaxation measurements to investigate the dynamics of the main chain of the chimera and showed that the fluctuating loop of the chimera is as rigid as three major helices. When we analyzed the chemical shift deviations and backbone HN-H-alpha scalar coupling constants, we found that the chimera showed an alpha-helical tendency in residues around the fluctuating loop. Our results suggest that the replacement of a highly fluctuating loop in a protein with a rigid structural element in a homologous one may be useful to stabilize the protein structure.
  • M Mizuguchi, Y Kobashigawa, Y Kumaki, M Demura, K Kawano, K Nitta
    PROTEINS-STRUCTURE FUNCTION AND GENETICS 49 1 95 - 103 2002年10月 [査読有り][通常論文]
     
    The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine a-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin. (C) 2002 Wiley-Liss, Inc.
  • A Matsuura, M Yao, T Aizawa, N Koganesawa, K Masaki, M Miyazawa, M Demura, Tanaka, I, K Kawano, K Nitta
    BIOCHEMISTRY 41 40 12086 - 12092 2002年10月 [査読有り][通常論文]
     
    Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.
  • T Suetake, T Aizawa, N Koganesawa, T Osaki, Y Kobashigawa, M Demura, S Kawabata, K Kawano, S Tsuda, K Nitta
    PROTEIN ENGINEERING 15 9 763 - 769 2002年09月 [査読有り][通常論文]
     
    Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The H-1-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, N-15-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone N-15 relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.
  • N Koganesawa, T Aizawa, H Shimojo, K Miura, A Ohnishi, M Demura, Y Hayakawa, K Nitta, K Kawano
    PROTEIN EXPRESSION AND PURIFICATION 25 3 416 - 425 2002年08月 [査読有り][通常論文]
     
    A small multifunctional cytokine, growth-blocking peptide (GBP), front the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem. we utilized a high-density fermentation method. The pH Of the medium in the fermenter was kept at 3.0, and the medium was collected within 48 h post methanol shift to minimize exposure of the target peptide to proteases, Recombinant GBP was purified through three reverse-phase HPLC columns, We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar, between chemically synthesized GBP and purified recombinant GBP. Up to 50 mg GBP was recovered pet, 1 L of yeast culture supernatant. (C) 2002 Elsevier Science (USA). All rights reserved.
  • N Fujitani, S Kawabata, T Osaki, Y Kumaki, M Demura, K Nitta, K Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 26 23651 - 23657 2002年06月 [査読有り][通常論文]
     
    The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.
  • M Sato, T Kanamori, N Kamo, M Demura, K Nitta
    BIOCHEMISTRY 41 7 2452 - 2458 2002年02月 [査読有り][通常論文]
     
    pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N-3(-), which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degreesC was 260s(-1) with in apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (0 --> hR' reaction) reported previously [Varo et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.
  • Functional Silk Proteins: Molecular Structure and Application to Biomaterials
    M. Demura, J. H. Yeo, K.G. Lee, Y.W. Lee
    Int. J. Industrial Entomology 4 1 - 4 2002年 [査読有り]
  • M Demura, M Noda, T Kajimoto, T Uchiyama, K Umemoto, CH Wong, T Asakura
    JOURNAL OF MOLECULAR STRUCTURE 602 215 - 222 2002年01月 [査読有り][通常論文]
     
    A structure of the peptidic mimic of sialyl Lewis X (Sle(x)) (alpha-N-acetyl-neuraminyl-(2,3)-beta-D-galactopyranosyl-(1,4)-[alpha-L-fucopyranosyl-(1,3)-beta-D-N-acetyl-glucosamine]) in an aqueous solution was studied using two-dimensional H-1 NNM spectroscopy. Complete assignments of H-1 NMR chemical shift of the SLe(x) mimic have been performed. The presence of three conformers of the SLex mimic in a solution was proposed by using distance geometry calculation based on NOE constraints, which were obtained from NOESY experiments. In addition, intermolecular interaction between the mimic and the crystal structure of E-selectin was refined using molecular dynamics. This suggested the conformational rearrangement of the functional groups of the conformers to the active sites of E-selectin. The relationship between the binding activities toward E-selectin and the three-dimensional structures of other mimics was also discussed. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Takashi Miyazaki, Tatsuo Kaneko, Jian Ping Gong, Yoshihito Osada, Makoto Demura, Makoto Suzuki
    Langmuir 18 4 965 - 967 2002年01月 [査読有り][通常論文]
  • T Koshiba, Y Kobashigawa, M Demura, K Nitta
    PROTEIN ENGINEERING 14 12 967 - 974 2001年12月 [査読有り][通常論文]
     
    Thermodynamics of thermal transitions of a calcium-binding lysozyme, canine milk lysozyme (CML), was studied using differential scanning calorimetry and compared with those for homologous proteins, human alpha-lactalbumin (alpha-hLA) and equine milk lysozyme (EML). The results showed that CML and EML exhibit two clear heat absorption peaks in the absence of calcium ions (apo-form), although the cooperative thermal transition of alpha-hLA is apparently absent in this form. The first peak represents the unfolding transition from the native to an unfolding intermediate state (N-I transition) and the second peak represents that from the intermediate to the thermally unfolded state (I-U transition). We interpret that the cooperative thermal transition, which is observed between the intermediate and the thermally unfolded states of CML and EML, comes from the native-like packing interaction in their intermediate states. Furthermore, to examine the role of the stabilization mechanism of CML intermediate, we constructed four variant CMLs (H21G, I56L, A93S and V109K), in which the residues of CML are substituted for those of EML, and also investigated their thermal stability. Especially the His21 and Val109 of CML play a role in stabilization of the intermediate state and their contributions to the unfolding free energy are estimated to be 2.0 and 1.8 kJ/mol, respectively. From the results of the mutational analysis, a few differences in the local helical interactions within the alpha-domain are found to be predominant in stabilizing the intermediate state.
  • T Aizawa, Y Hayakawa, A Ohnishi, N Fujitani, KD Clark, MR Strand, K Miura, N Koganesawa, Y Kumaki, M Demura, K Nitta, K Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 34 31813 - 31818 2001年08月 [査読有り][通常論文]
     
    Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta -sheet (Tyr(11)Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.
  • T Niidome, H Murakami, M Kawazoe, T Hatakeyama, Y Kobashigawa, M Matsushita, Y Kumaki, M Demura, K Nitta, H Aoyagi
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 11 14 1893 - 1896 2001年07月 [査読有り][通常論文]
     
    We have designed and synthesized of carbohydrate-binding peptides. gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of (1)H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide. [Gln(1.1 '),Trp(3.3 ')]-gramisidin S and mannose were observed. indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues. (C) 2001 Elsevier Science Ltd. All rights reserved.
  • Y Kurokawa, N Koganesawa, Y Kobashigawa, T Koshiba, M Demura, K Nitta
    JOURNAL OF PROTEIN CHEMISTRY 20 4 293 - 303 2001年05月 [査読有り][通常論文]
     
    Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) approximate to SS3(Cys65-Cys81) > SS4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D-helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.
  • Y Kobashigawa, K Miura, M Demura, N Nemoto, T Koshiba, K Nitta, S Tsuda
    JOURNAL OF BIOMOLECULAR NMR 19 4 387 - 388 2001年04月 [査読有り][通常論文]
  • Y Hori, M Demura, M Iwadate, AS Ulrich, T Niidome, H Aoyagi, T Asakura
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 2 302 - 309 2001年01月 [査読有り][通常論文]
     
    Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using H-1-NMR spectroscopy in perdeuterated (SDS-d(25)) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha -helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state H-2-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d(4)) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha -segments and beta -segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically N-15-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state N-15-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.
  • Y Kobashigawa, M Demura, T Koshiba, Y Kumaki, K Kuwajima, K Nitta
    PROTEINS-STRUCTURE FUNCTION AND GENETICS 40 4 579 - 589 2000年09月 [査読有り][通常論文]
     
    The native state H-1, N-15 resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states, On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state. (C) 2000 Wiley-Liss, Inc.
  • JD van Beek, L Beaulieu, H Schafer, M Demura, T Asakura, BH Meier
    NATURE 405 6790 1077 - 1079 2000年06月 [査読有り][通常論文]
     
    Silks are fibrous proteins that form heterogeneous, semi-crystalline solids. Silk proteins have a variety of physical properties reflecting their range of functions. Spider dragline silk, for example, has high tensile strength and elasticity(1), whereas other silks(2) are better suited to making housing, egg sacs or the capture spiral of spiders' webs. The differing physical properties arise from variation in the protein's primary and secondary structure, and their packing in the solid phase. The high mechanical performance of spider dragline silk, for example, is probably due to a beta-sheet conformation of poly-alanine domains(3), embedded as small crystallites within the fibre. Only limited structural information can be obtained from diffraction of silks(3-6), so further characterization requires spectroscopic studies such as NMR7-11. However, the classical approach to NMR structure determination(12) fails because the high molecular weight(13), repetitive primary structure(13) and structural heterogeneity of solid silk means that signals from individual amino-acid residues cannot be resolved. Here we adapt a recently developed solid-state NMR technique(14,15) to determine torsion angle pairs (phi, Psi) in the protein backbone, and we study the distribution of conformations in silk from the Eri silkworm, Samia cynthia ricini. Although the most probable conformation in native fibres is an anti-parallel beta-sheet, film produced from liquid directly extracted from the silk glands appears to be primarily alpha-helical.
  • M Mizuguchi, K Masaki, M Demura, K Nitta
    JOURNAL OF MOLECULAR BIOLOGY 298 5 985 - 995 2000年05月 [査読有り][通常論文]
     
    The molten globule state of alpha-lactalbumin has ordered secondary structure in the alpha-domain, which comprises residues 1 to 34 and 86 to 123. Tn order to investigate which part of a polypeptide is important for stabilizing the molten globule state of alpha-lactalbumin, we have produced and studied three chimeric proteins of bovine and human alpha-lactalbumin. The stability of the molten globule state formed by domain-exchanged alpha-lactalbumin, in which the amino acid sequence in the alpha-domain comes from human alpha-lactalbumin and that in the beta-domain comes from bovine alpha-lactalbumin, is the same as that of human alpha-lactalbumin and is substantially greater than that of bovine alpha-lactalbumin. Therefore, our results show that the stability of the molten globule state of alpha-lactalbumin is determined by the alpha-domain and the beta-domain is not important for stabilizing the molten globule state. The substitution of residues 1 to 34 of bovine alpha-lactalbumin with those of human alpha-lactalbumin substantially increases the stability of the molten globule state, while the substitution of residues 86 to 123 of bovine alpha-lactalbumin with those of human alpha-lactalbumin decreases the stability of the molten globule state. Therefore, residues 1 to 34 in human alpha-lactalbumin is more important for the stability of the human alpha-lactalbumin molten globule state than residues 86 to 123. The stabilization of the molten globule state due to substitution of both residues 1 to 34 and 86 to 123 is not identical with the sum of the two individual substitutions, demonstrating the non-additivity of the stabilization of the molten globule state. This result indicates that there is a long-range interaction between residues 1 to 34 and 86 to 123 in the molten globule state of human alpha-lactalbumin. The differences in the stabilities of the molten globule states are well correlated with the averaged helical propensity values in the alpha-domain when the long-range interactions are negligible, suggesting that the local interaction is the dominant term for determining the stability of the molten globule state. Our results also indicate that the apparent cooperativity is closely linked to the stability of the molten globule state, even if the molten globule state is weakly cooperative. (C) 2000 Academic Press.
  • K Sasahara, M Demura, K Nitta
    BIOCHEMISTRY 39 21 6475 - 6482 2000年05月 [査読有り][通常論文]
     
    Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.
  • T Koshiba, M Yao, Y Kobashigawa, M Demura, A Nakagawa, Tanaka, I, K Kuwajima, K Nitta
    BIOCHEMISTRY 39 12 3248 - 3257 2000年03月 [査読有り][通常論文]
     
    Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 Angstrom to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the H-1 NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 OC) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.
  • M Osanai, M Okudaira, J Naito, M Demura, T Asakura
    INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 30 3 225 - 232 2000年03月 [査読有り][通常論文]
     
    The derivation of alanine in fibroin was investigated using NMR and selective isotopic labelling. (H2O)-H-2 infused orally into 5th instar larvae was incorporated into the proton of the methyl group of alanine in fibroin. Proton exchange among alanine, glycine and serine was also found. Incorporation of C-13 from [2-C-13]acetate into alanine C2 and C3 and glycine C2 in fibroin, and also C4 of free glutamine plus glutamate was observed in vivo. Hemolymph contained a peak for C4 of glutamate plus glutamine, and an alanine C3 peak appeared transiently. Thus, it is suggested that the C-skeleton of alanine formed was derived from L-malate via the TCA-cycle, and that this alanine is utilized in part for fibroin synthesis. Spectra of the hemolymph extract of larvae infused orally with [N-15(2)]urea showed no 1(5N)-compounds, whereas those of larvae injected subcutaneously showed only one peak of urea, whose intensity decreased with time, as shown in the in vivo spectra of a living larva infused with [N-15(2)]urea. The solution NMR spectrum of fibroin showed no N-15-labelled compounds. Temporal changes in the peak intensities of six compounds in the spectra of a Living larva infused with [N-15]ammonium demonstrated a process in which N-15 was incorporated into fibroin containing N-15-alanine through the amide group of glutamine and the amino group of glutamate. Thus, alanine biosynthesis from the. TCA-cycle originates mainly from water, L-malate and ammonium. The fact that no N-15-urea was detected in the hemolymph extract of larvae infused with [N-15]ammonium suggests that N-15-urea found in the above in vivo spectra may be that accumulated in the hindgut. Thus, excess ammonium in the body causes the production of urea by the urea-cycle. In Samia larvae, urea was not reutilized but excreted. The metabolic relationships between the assimilation of ammonium and the function of the urea-cycle are discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.
  • Takuro Ito, Yoshitsugu Maruhashi, Makoto Demura, Tetsuo Asakura
    Polymer 41 3 859 - 866 2000年02月
  • T Asakura, M Demura
    MACROMOLECULAR SYMPOSIA 143 1 - 10 1999年08月 [査読有り][通常論文]
     
    [1-C-13]Gly, L-[1-C-13]Ala, [N-15]Gly, L-[N-15]Ala, [2,2-H-2(2)]Gly, L-[3,3-H-2(2)]Ser and [3,3,3-H-2(3)]Ala labeled silk fibroin fibers from Bombyx mori and Samia cynthia ricini silkworms were prepared in order to analyze structure of backbone and dynamics of side chain. The torsion angles phi and psi were determined from the angular dependent C-13 and N-15 solid State NMR spectra for uniaxially oriented fiber samples. In addition, the characteristic side chain dynamics of Ser residue determined from solid state H-2 NMR measurements was compared with those of Ala and Gly residues.
  • Y Hori, M Demura, T Niidome, H Aoyagi, T Asakura
    FEBS LETTERS 455 3 228 - 232 1999年07月 [査読有り][通常論文]
     
    Solid state P-31 NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The P-31 chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group, Macroscopically oriented MP-lipid samples between glass plates were studied by P-31 NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes. (C) 1999 Federation of European Biochemical Societies.
  • T Asakura, M Iwadate, M Demura, MP Williamson
    INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 24 2-3 167 - 171 1999年03月 [査読有り][通常論文]
     
    The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the C-13 chemical shift and local structure in proteins. To determine this relationship,C-13 chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and C-13 NMR chemical shift data in aqueous solution reported for 40 proteins. The C-13 CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with C-13 CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the C-13 chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their C-13 isotropic NMR chemical shifts in the solid state. There are two area of the phi,Psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the C-13 chemical shift contour plots. (C) 1999 Elsevier Science B.V. All rights reserved.
  • A Higuchi, S Uchiyama, M Demura, T Asakura, CS Cho, T Akaike, H Takarada, M Hara
    CYTOTECHNOLOGY 31 3 233 - 242 1999年 [査読有り][通常論文]
     
    Human colorectal adenocarcinoma tumor (CW2) cells were cultivated in RPMI 1640 media containing 0-7.5 mM aspirin and 10% fetal bovine serum for the production of carcinoembryonic antigen (CEA). By adding aspirin to the media, the production of CEA per cell increased by up to one hundred fold compared to cultivation in normal media containing no aspirin, even though the total cell concentration decreased with the increase in aspirin in the media. The production of CEA was also investigated for CW2 cells cultured on silk fibroin, poly(gamma-benzyl-L-glutamate) and poly(gamma-benzyl-L-glutamate)/poly(ethylene oxide) diblock copolymer films prepared by the Langmuir-Blodgett and casting methods. The highest production of CEA per cell was observed for the CW2 cells on poly(gamma-benzyl-L-glutamate) and its diblock copolymer films prepared by the Langmuir-Blodgett method in the medium containing 5 mM aspirin after 168 hr of inoculation. This originates from the fact that the cell density on the films in the medium containing 5 mM aspirin was the lowest under these conditions. It is suggested that CW2 cells produce CEA more effectively when the cell growth is suppressed by addition of toxic chemicals such as aspirin or by culture on unfavorable films for cell growth.
  • Tetsuo Asakura, Yasunobu Yamazaki, Koo Wey Seng, Makoto Demura
    Journal of Molecular Structure 446 3 179 - 190 1998年05月
  • M Demura, M Minami, T Asakura, TA Cross
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 120 6 1300 - 1308 1998年02月 [査読有り][通常論文]
     
    Isotopic labeling of Bombyx mori silk fibroin was achieved biosynthetically using two approaches. First, labeled fibroin was achieved by feeding silk worms [C-13(1)]Gly and [C-13(1)]Ala along with an artificial diet. Second, in vitro production of [N-15]Gly and [N-15]Ala labeled B. mori silk fibroin was accomplished by culturing the posterior silk glands isolated from five-day-old silkworm larvae in the fifth instar stage. Orientation-dependent N-15 and C-13 solid-state NMR spectra of these isotope-labeled silk fibroin fibers were observed when the fiber axis was arranged at various angles between 0 degrees and 90 degrees to the magnetic field direction. Such data from the silk II structure was simulated on the basis of the chemical shift anisotropy to determine the Euler angles that relate the principal axis system (PAS) to the fiber axis coordinate system. The dipolar modulated N-15 and C-13 chemical shift powder pattern spectra of C-13-N-15 double-labeled silk fibroin model peptides were observed and simulated to determine the Euler angles for transforming the PAS to molecular symmetry axis (MSA) system. The specific orientations of N-H, N-C-1, C-1=O, and C-1-N bonds for Ala and Gly residues in the oriented silk fibroin fiber were determined with a combination of these Euler angles. The conformational space for the Ala and Gly residues of the silk fibroin fibers was substantially reduced with these bond orientations and the known Calpha(i-1)-Calpha(i+1) vector orientation from fiber diffraction studies. The best fit torsion angles (phi,psi) within the reduced conformational space were determined as (-140 degrees, 142 degrees) and (-139 degrees, 135 degrees), respectively within experimental error (+/-5 degrees). The distance of the unit cell length determined here results in excellent agreement with the fiber diffraction data.
  • Makoto Demura, Masashi Minami, Tetsuo Asakura, T. A. Cross
    Journal of the American Chemical Society 120 6 1300 - 1308 1998年01月27日
  • Makoto Demura, Masashi Minami, Tetsuo Asakura, T. A. Cross
    Journal of the American Chemical Society 120 6 1300 - 1308 1998年01月27日
  • M Demura, Y Yamazaki, T Asakura, K Ogawa
    JOURNAL OF MOLECULAR STRUCTURE 441 2-3 155 - 163 1998年01月 [査読有り][通常論文]
     
    Carbon-13 isotopic labeling of B. mori silk fibroin was achieved biosynthetically with [I-C-13] glycine in order to determine the carbonyl bond orientation angle of glycine sites with the silk fibroin. Angular dependence of C-13 solid State NMR spectra of uniaxially oriented silk fibroin fiber block sample due to the carbonyl C-13 chemical shift anisotropy was simulated according to the chemical shift transformation with Euler angles, alpha(F) and beta(F), from principal axis system (PAS) to fiber axis system (FAS). The another Euler angles, alpha(DCO) and beta(DCO), for transformation from PAS to the molecular symmetry axis were determined from the [1-C-13] glycine sequence model compounds for the silk fibroin. By the combination of these Euler angles, the carbonyl bond orientation angle with respect to FAS of the [1-C-13] glycine sites of the silk fibroin was determined to be 90 +/- 5 degrees. This value is in agreement with the X-ray diffraction and our previous solid state NMR data of B. mori silk fibroin fiber (a typical beta-pleated sheet) within experimental error. (C) 1998 Elsevier Science B.V.
  • Tetsuo Asakura, Masashi Minami, Reiko Shimada, Makoto Demura, Minoru Osanai, Teruaki Fujito, Mamoru Imanari, Anne S. Ulrich
    Macromolecules 30 8 2429 - 2435 1997年04月01日
  • NMR Study of Silk I Structure of Bombyx mori Silk Fibroin with 15N and 13C NMR Chemical Shift Contour Plots
    T. Asakura, M. Demura, T. Date, M. Miyashita, K. Ogawa, M. P. Williamson
    Biopolymers 41 193 - 203 1997年 [査読有り]
  • Tetsuo Asakura, Takehito Konakazawa, Makoto Demura, Takuro Ito, Yoshitsugu Maruhashi
    Polymer 37 10 1965 - 1973 1996年05月
  • Refinement of the Solution Structure of Neurokinin A (4-10) on the Basis of 1H NMR Chemical Shift Calculations
    T. Asakura, M. Iwadate, T. Date, M. Demura, K. Nokihara
    Peptide Chem 345 - 348 1996年 [査読有り]
  • High-Resolution Solid State 13C NMR Spectroscopy of Syndiotactic Polypropylene
    T. Asakura, A. Aoki, T. Date, M. Demura
    Polymer J 28 24 - 29 1996年
  • The Relationship between Amide Proton Chemical Shifts and Secondary Structure in Proteins
    T. Asakura, K. Taoka, M. Demura, M. P. Williamson
    J. Biomol. NMR 6 227 - 236 1995年 [査読有り]
  • Structural Characterization of Samia cynthia ricini Silk Fibroin as Biomaterial
    M. Demura, T. Asakura
    Int. J. Wild Silkmoth & Silk 1 139 - 141 1994年 [査読有り]
  • Structure of Bombyx mori Silk Fibroin Studied by REDOR NMR Spectroscopy
    T. Asakura, A. Aoki, M. Demura, J. M. Joers, R. C. Rosanske, T. Gullion
    Polymer J 26 1405 - 1408 1994年 [査読有り]
  • Structural analysis of highly oriented poly (P-phenylene-ferephthalamide) by 15N solid-state nuclear magnetic resonance
    J.-H. Yeo, M. Demura, T. Asakura, T. Fujito, M. Imanari, L. K. Nicholson, T. A. Cross
    Solid State Nuclear Magnetic Resonance 3 209 - 218 1994年 [査読有り]
  • A Study on the Hydration of Bombyx mori Silk Fibroin by Nuclear Magnetic Resonance Spectroscopy
    M. Minami, R. Takatsu, M. Demura, T. Asakura
    Sen-i Gakkaishi 50 498 - 504 1994年 [査読有り]
  • Determination of the Structure of [1-13C] Glycine-[15N] Alanine Double Labeled Bombyx mori Silk Fibroin Fibers Using Solid State 15N NMR
    T. Asakura, M. Demura, Y. Hiraishi, K. Ogawa, A. Uyama
    Chem. Lett. 2249 - 2252 1994年 [査読有り]
  • Activation Energy for Permeation of Phoshonium Cations Through Phospholipid Bilayer Membrane
    A. Ono, S. Miyauchi, M. Demura, T. Asakura, N. Kamo
    Biochemistry 33 4312 - 4318 1994年 [査読有り]
  • A Method for Studying the Structure of Uniaxially Aligned Biopolymers Using Solid State 15N NMR: Application to Bombyx mori Silk Fibroin Fibers
    L. K. Nicholson, T. Asakura, M. Demura, T. A. Cross
    Biopolymers 33 847 - 861 1993年 [査読有り]
  • Structural Analysis of Uniaxially Aligned Polymers Using Solid State 15N NMR
    Asakura, J.-H. Yeo, M. Demura, T. Itoh, T. Fujito, M. Imanari, L. K. Nicholson, T. A. Cross
    Macromolecules 26 6660 - 6663 1993年 [査読有り]
  • T ASAKURA, R SAKAGUCHI, M DEMURA, T MANABE, A UYAMA, K OGAWA, M OSANAI
    BIOTECHNOLOGY AND BIOENGINEERING 41 2 245 - 252 1993年01月 [査読有り][通常論文]
     
    An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient N-15 isotope labeling of silk fibroin, which is required for N-15 solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system.
  • T ASAKURA, N NAKAYAMA, M DEMURA, A ASANO
    MACROMOLECULES 25 19 4876 - 4881 1992年09月 [査読有り][通常論文]
     
    The C-13 NMR spectrum of regioirregular polypropylene containing up to 40 mol % of inverted propylene units was assigned using the 2D INADEQUATE technique, the INEPT technique, and C-13 chemical shift calculations based on the C-13 NMR gamma-effect and the application of the rotational isomeric state model and also on the additive rules for C-13 chemical shifts of methyl-substituted alkanes. The presence of four kinds of sequences containing head-to-head and/or tail-to-tail units in the sample was confirmed. The C-13-C-13 connectivities of the 2D INADEQUATE spectrum were extremely useful in the assignment of overlapping peaks as well as the identification of several such sequences. The peaks were assigned to each carbon in these sequences by taking tacticity into account. There is exclusively a meso head-to-head unit in the sequence where a single inverted unit is contained in several head-to-tail units.
  • T ASAKURA, M KITAGUCHI, M DEMURA, H SAKAI, K KOMATSU
    JOURNAL OF APPLIED POLYMER SCIENCE 46 1 49 - 53 1992年09月 [査読有り][通常論文]
     
    The enzyme glucose oxidase (GOD) was immobilized on the nonwoven fabrics, which have excellent properties in diffusivity of substrates, mechanical strength, and handling, with Bombyx mori silk fibroin gel. The nonwoven fabrics of silk fibroin, viscose rayon, polyethyleneterephthalate, 6-nylon, and polypropylene with activated surface by fluoline treatment were used. The stabilities of GOD to heat or pH changes were much improved by the immobilization as well as the case of the GOD immobilized in the silk fibroin membrane. Among nonwoven fabrics, silk fibroin was the most excellent support material for the immobilization of GOD although all nonwoven fabrics used here are able to be used as the support materials. The increase of the sensitivity was observed when the glucose sensor was made with the GOD immobilized on nonwoven silk fabrics as four times compared with the case of the GOD immobilized in the silk fibroin membrane.
  • T ASAKURA, M DEMURA, Y WATANABE, K SATO
    JOURNAL OF POLYMER SCIENCE PART B-POLYMER PHYSICS 30 7 693 - 699 1992年06月 [査読有り][通常論文]
     
    The dynamical behavior of the Bombyx mori silk fibroin chain and of absorbed water in silk fiber, film, and powder has been studied by H-1 pulsed nuclear magnetic resonance (NMR). Segmental motions do not occur and only the rapid rotation of the methyl groups of alanine residues is observed from -120 to 130-degrees-C. This is independent of the conformation or form of the silk fibroin samples. Magnetization of dry silk fibroin by the solid-echo method shows a single Gaussian decay, while two components are observed in the solid-echo signals of films containing 6-10 w/w% water. An immobile component with a T2 value of 11-mu-s is attributed to silk fibroin, and the mobile component to bound water. The T2 of the latter varies from 50 to 200-mu-s, depending on the sample. The dynamical behavior of water trapped in the film is discussed on the basis of these T2 values.
  • M DEMURA, T TAKEKAWA, T ASAKURA, A NISHIKAWA
    BIOMATERIALS 13 5 276 - 280 1992年 [査読有り][通常論文]
     
    Bombyx mori silk fibroin fabrics were treated with low-temperature-plasma using various gases. Alkaline phosphatase was immobilized onto the fabrics using the CNBr method. The enzyme activity was much improved by plasma treatment, especially when both O2 and CF4 were used. Using the ESCA technique it was found that the activities of the immobilized enzyme were strongly correlated with the C1s peak intensities of the atomic group -C-O-CF3. The apparent Michaelis constant for the enzyme decreased with increasing amounts of -C-O-CF3.
  • MP WILLIAMSON, T ASAKURA, E NAKAMURA, M DEMURA
    JOURNAL OF BIOMOLECULAR NMR 2 1 83 - 98 1992年01月 [査読有り][通常論文]
     
    The chemical shifts of C(alpha)H protons have been calculated for 9 proteins, based on coordinates taken from high-resolution crystal structures. Chemical shifts were calculated using ring-current shifts, shifts arising from magnetic anisotropies of bonds. and shifts arising from the polarizing effect of polar atoms on the C(alpha)-H bond. The parameters used were refined iteratively to give the best fit to (experimental - random coil) shifts over the set of 9 proteins. A further small correction was made to the averaged Gly C(alpha)H shift. The calculated shifts match observed shifts with correlation coefficients varying between 0.45 and 0.86, with a standard deviation of about 0.3 ppm. The differences between calculated and observed shifts have been studied in detail, including an analysis of different crystal structures of the same protein, and indicate that most of the differences can be accounted for by small differences between the structure in solution and in the crystal. Calculations using NMR-derived structures give a poor fit. The calculations reproduce the experimentally observed differences between chemical shifts for C(alpha)H in alpha-helix and beta-sheet. Most of thy differentiation in secondary-structure-dependent shifts arises from electric field effects, although magnetic anisotropy also makes a large contribution to the net shift. Applications of the calculations to assignment (including stereospecific assignment) and structure determination are discussed.
  • Porous Membrane of Bombyx mori Silk Fibroin: Structure Characterization, Physical Properties and Application to Glucose Oxidase- Immobilization
    M. Demura, T. Asakura
    J. Membrane Sci. 59 39 - 52 1991年 [査読有り]
  • Membrane Potential of Bombyx mori Silk Fibroin Membrane Induced by an Immobilized Enzyme Reaction
    M. Demura, T. Komura, T. Asakura
    Bioelectrochem. Bioenerg. 26 167 - 175 1991年 [査読有り]
  • Metabolic Flux and Incorporation of [2-13C] Glycine into Silk Fibroin Studied by 13C NMR in vivo and in vitro
    T. Asakura, M. Demura, M. Nagashima, R. Sakaguchi, M. Osanai, K. Ogawa
    Insect Biochem. 21 743 - 748 1991年 [査読有り]
  • NMR Imaging of Diffusion of Small Organic Molecules in Silk Fibroin Gel
    T. Asakura, M. Demura, H. Ogawa, K. Matsushita, M. Imanari
    Macromolecules 24 620 - 622 1991年 [査読有り]
  • Ring-Current Effects and Magnetic Anisotropy Effects of Carbonyl Groups on the α-CH Proton Chemical Shifts of the Basic Pancreatic Trypsin Inhibitor and Tendamistat
    T. Asakura, E. Nakamura, H. Asakawa, M. Demura
    J. Magn. Reson. 93 355 - 360 1991年 [査読有り]
  • Carbon-13 NMR Spectral Assignment of Five Polyolefins Determined from the Chemical Shift Calculation and the Polymerization Mechanism
    T. Asakura, M. Demura, Y. Nishiyama
    Macromolecules 24 2334 - 2340 1991年 [査読有り]
  • 2D-INADEQUATE 13C NMR Spectra of Atactic Polyolefins
    T. Asakura, K. Hirano, M. Demura, K. Kato
    Makromol. Chem. Rapid Commun 12 215 - 220 1991年 [査読有り]
  • S LENKA, M DEMURA, T ASAKURA
    MAKROMOLEKULARE CHEMIE-MACROMOLECULAR CHEMISTRY AND PHYSICS 191 6 1321 - 1327 1990年06月 [査読有り][通常論文]
  • T ASAKURA, H YAMADA, M DEMURA, M OSANAI
    INSECT BIOCHEMISTRY 20 3 261 - 266 1990年 [査読有り][通常論文]
  • M DEMURA, T ASAKURA, E NAKAMURA, H TAMURA
    JOURNAL OF BIOTECHNOLOGY 10 2 113 - 119 1989年05月 [査読有り][通常論文]
  • Naoki Kamo, Makoto Demura, Yonosuke Kobatake
    Journal of Electroanalytical Chemistry 275 1 33 - 46 1989年 [査読有り][通常論文]
     
    Membrane potential measurements of small cells or vesicles which are too small to use microelectrodes are performed with the use of a lipophilic ion as a probe. Although the principle of this method is simple and clear, adsorption of probes to membranes and/or intracellular constituents causes errors. On the basis of the binding model proposed previously and with the use of data on the binding to intact mitochondria, the relation between the true and the observed membrane potentially (called the uncorrected value) is calculated and a linear calibration equation, (true membrane potential) = A × (uncorrected value) + B, is found to hold for mitochondria in the range from -100 to -200 mV (determination coefficient > 0.99 for all cases). Here, membrane potentials are defined with respect to the outside. The values of A and B depend on the initial probe concentration, co. But for TPMP+ (triphenylmethylphosphonium), the dependence on co is small, and this fact has been shown experimentally. On the other hand, for TPP+ (tetraphenylphosphonium) the dependence is evident. For TPMP+, A and B do not depend on the protein concentration while for TPP+, they do depend on it. The values of the inner volume change A and B. Fortunately, actual calculation of the membrane potential is not affected very much by the value of volume employed, especially when dilute TPP+ is used. This type of calibration equation has been obtained experimentally by other authors, and is very useful from a practical point of view. The equation obtained in this study agrees well with that obtained experimentally by other authors for mitochondria suspended in a medium of high ionic strength, although the medium was not strictly identical. They considered the value obtained with valinomycin + Rb+ to be the true membrane potential. © 1989.
  • M DEMURA, T ASAKURA, T KUROO
    BIOSENSORS 4 6 361 - 372 1989年 [査読有り][通常論文]
  • M DEMURA, T ASAKURA
    BIOTECHNOLOGY AND BIOENGINEERING 33 5 598 - 603 1989年01月 [査読有り][通常論文]
  • T ASAKURA, J KANETAKE, M DEMURA
    POLYMER-PLASTICS TECHNOLOGY AND ENGINEERING 28 4 453 - 469 1989年 [査読有り][通常論文]
  • Application of silk fibroin membrane; Relationship between membrane preparation, membrane potential and ion permeability
    A. Kitamura, A. Shibamoto, M. Demura
    Sen-i Gakkaishi 44 193 - 198 1988年 [査読有り]
  • 23Na and 27Al NMR Studies of the interaction between Bombyx mori silk fibroin and metal ions trapped in the porous silk fibroin membrane
    T. Asakura, M. Demura, M. Tsutsumi
    Makromol. Chem. Rapid Commun. 9 835 - 839 1988年 [査読有り]
  • NMR of Silk Fibroin 11. 1H NMR Analysis of Water Orientation in Porous Silk Fibroin Membrane
    T. Asakura, M. Demura
    Sen-i Gakkaishi 44 535 - 540 1988年 [査読有り]
  • Water Sorption, Membrane Potential and Ion Permeability of Styrene Grafted Bombyx mori Silk Fibroin Membrane
    M. Demura, A. Kitamura, A. Shibamoto, T. Asakura
    J. Appl. Polym. Sci. 36 535 - 543 1988年 [査読有り]
  • 13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae
    T. Asakura, Y. Kawaguchi, M. Demura, M. Osanai
    Insect Biochem. 18 531 - 538 1988年 [査読有り]
  • Makoto Demura, Naoki Kamo, Yonosuke Kobatake
    BIOCHIMICA ET BIOPHYSICA ACTA 894 3 355 - 364 1987年12月17日 [査読有り][通常論文]
     
    Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and / or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 μM) and low (under 20 μM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25°C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the Langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 μM. © 1987.
  • Makoto Demura, Naoki Kamo, Yonosuke Kobatake
    BIOCHIMICA ET BIOPHYSICA ACTA 903 2 303 - 308 1987年10月02日 [査読有り][通常論文]
     
    Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+. © 1987.
  • Polymerization Mechanism and Conformation of Poly(1-butene)
    T. Asakura, M. Demura, K. Yamamoto, R. Chujo
    Polymer 28 1037 - 1040 1987年 [査読有り]
  • N KAMO, M DEMURA, Y KOBATAKE
    JOURNAL OF MEMBRANE SCIENCE 27 2 233 - 239 1986年06月 [査読有り][通常論文]
  • M DEMURA, N KAMO, Y KOBATAKE
    BIOELECTROCHEMISTRY AND BIOENERGETICS 14 4-6 439 - 448 1985年12月 [査読有り][通常論文]
  • Makoto Demura, Naoki Kamo, Yonosuke Kobatake
    BIOCHIMICA ET BIOPHYSICA ACTA 820 2 207 - 215 1985年11月07日 [査読有り][通常論文]
     
    The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of Ciapp Co for various probes are plotted against the binding coefficient. Here, Ciapp is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential. © 1985.
  • Makoto Demura, Naoki Kamo, Yonosuke Kobatake
    BIOCHIMICA ET BIOPHYSICA ACTA 812 2 377 - 386 1985年01月25日 [査読有り][通常論文]
     
    The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-5) and tetraphenylphosphonium (TPP+). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP+, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined. © 1985.

書籍

  • クロスメディアHR総合研究所 (担当:分担執筆範囲:北海道大学:持続可能な社会の創り手として、社会に貢献する)
    クロスメディア・パブリッシング,インプレス (発売) 2021年04月 (ISBN: 9784295404507) 182p
  • 光で働く微生物のロドプシン
    塚本卓, 出村誠 (担当:共著)
    化学・化学同人 2021年04月
  • シルクと持続可能な開発目標(SDGs)
    出村 誠 (担当:単著)
    繊維機械学会誌『せんい』, 73(2) 115-120 2020年
  • 持続可能な開発目標(SDGs)とシルク繊維、繊維機械学会誌『せんい』, Vol.73
    出村 誠 (担当:単著範囲:巻頭言)
    一般社団法人 日本繊維機械学会 2020年
  • タンパク質、『基礎高分子科学』改訂版・９章 生体高分子
    出村誠、朝倉哲郎 (担当:共著)
    東京化学同人 2020年
  • カイコ絹糸の模倣からイノベーションへ, 特集 生物機能模倣技術, アグリバイオ
    出村 誠 (担当:単著範囲:アグリバイオ, vol.1, 235-239)
    北隆館 2017年03月
  • 光のエネルギー利用27 ハロロドプシン『光と生命の事典』（日本光生物学協会「光と生命の事典」編集委員会 編）
    出村 誠 (担当:分担執筆範囲:光のエネルギー利用27 ハロロドプシン)
    朝倉書店 2016年02月
  • Photochemistry of Halorhodopsin in Optogenetics
    Takashi Kikukawa, Naoki Kamo, Makoto Demura (担当:共著範囲:Photochemistry of Halorhodopsin)
    Springer 2015年
  • オプトジェネティクス（光遺伝学）～光操作による行動制御技術～
    出村 誠 (担当:共著範囲:ハロロドプシンの構造と分子機能解析)
    エヌ・ティー・エス 2013年
  • 広がるNMRの世界 -40人の研究者からの熱いメッセージ-
    出村 誠 (担当:共著範囲:ペプチドの機能と構造：最小へリックスの暗号)
    コロナ社 2011年
  • ペプチドの機能と構造：最小へリックスの暗号、『広がるNMRの世界 -40人の研究者からの熱いメッセージ-』
    コロナ社 2011年
  • Photo-induced Proton Transfers of Microbial Rhodopsins in Photochemistry
    InTech ISBN 979-953-307-364-3 2011年
  • NMR studies of protein folding: Folding studies of calcium-binding lysozyme and alpha-lactalbumin, Annual Reports on NMR Spectroscopy
    Springer 2009年
  • 基礎から学ぶ構造生物学
    出村誠, 神谷昌克, 相沢智康 (担当:共著範囲:光イオンポンプ)
    共立出版 2008年
  • バイオとナノの融合I新生命科学の基礎
    出村誠 (担当:共著範囲:光で機能するレチナール膜タンパク質のナノ構造)
    北海道大学出版会 2007年
  • 出村 誠 
    [北海道大学大学院先端生命化学研究院] 2007年
  • Modern Magnetic Resonance, Part I Application in Chemistry, Biological and Marine Sciences
    M. Demura (担当:共著範囲:NMR insight of structural stability and folding of calcium- binding lysozyme)
    Springers 2006年
  • 基礎高分子科学
    出村誠, 朝倉哲郎 (担当:共著範囲:タンパク質)
    東京化学同人 2006年

作品等

  • ひらめきときめきサイエンスの実施
    2011年

その他活動・業績

  • 塚本卓, 出村誠 化学 76 (4) 2021年
  • GU Hao, 加藤貴純, 石田博昭, 熊木康裕, 塚本卓, 塚本卓, 菊川峰志, 菊川峰志, 出村誠, 出村誠, VOGEL HANS J., 相沢智康, 相沢智康 Abstracts. Annual Meeting of the NMR Society of Japan 59th 2020年
  • 平峰里菜, 久米田博之, 久米田博之, 熊木康裕, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 中村公則, 綾部時芳, 相沢智康, 相沢智康 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.4J29a01 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  • 山野めぐみ, KUDDUS MD Ruhul, RUMI Farhana, 堤元佐, 久米田博之, 久米田博之, 熊木智康, 神谷昌克, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 相沢智康, 相沢智康 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.4J29a04 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  • 堀江裕紀子, 根本英幸, 池川繁男, 熊木康裕, 大西裕季, 久米田博之, 久米田博之, 出村誠, 出村誠, 相沢智康, 相沢智康 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.2A05p10 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  • 稲村勇雅, 久米田博之, 久米田博之, 熊木康裕, 大西裕季, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 小山朗夫, 伊東昌章, 岩波俊介, 相沢智康, 相沢智康 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.3A04p09 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  • 出村 誠 アグリバイオ 1 235 -239 2017年03月 [査読無し][招待有り]
  • 田巻初, 田巻初, 斉藤優太, 江川文子, 菊川峰志, 相沢智康, 出村誠, 藤原敏道 Abstracts. Annual Meeting of the NMR Society of Japan 55th 230‐231 2016年11月16日 [査読無し][通常論文]
  • 山野めぐみ, 神谷昌克, 熊木康裕, 菊川峰志, 出村誠, 相沢智康 日本農芸化学会大会講演要旨集(Web) 2016 4C001 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 田巻初, 斉藤優太, 江川文子, 菊川峰志, 神谷昌克, 相沢智康, 藤原敏道, 出村誠 Abstracts. Annual Meeting of the NMR Society of Japan 54th 236 -237 2015年11月06日 [査読無し][通常論文]
  • 若松瞭太, 神谷昌克, 相沢智康, 熊木康裕, 菊川峰志, 出村誠 Abstr Annu Meet NMR Soc Jpn 54th 156 -157 2015年11月06日 [査読無し][通常論文]
  • 斉藤優太, 田巻初, 江川文子, 菊川峰志, 神谷昌克, 相沢智康, 藤原敏道, 出村誠 Abstr Annu Meet NMR Soc Jpn 54th 234 -235 2015年11月06日 [査読無し][通常論文]
  • 柴崎宏介, 重村洋明, 菊川峰志, 神谷昌克, 相沢智康, 河野敬一, 加茂直樹, 出村誠 高圧討論会講演要旨集 55th 71 2014年11月10日 [査読無し][通常論文]
  • 比嘉一葉, 神谷昌克, 菊川峰志, 熊木康裕, 出村誠, 相沢智康 Abstr Annu Meet NMR Soc Jpn 53rd 234 -235 2014年11月04日 [査読無し][通常論文]
  • 柚原光佑, 神谷昌克, 熊木康裕, 滝谷重治, 菊川峰志, 河野敬一, 出村誠, 相沢智康 Abstr Annu Meet NMR Soc Jpn 53rd 244 -245 2014年11月04日 [査読無し][通常論文]
  • 田巻初, 江川文子, 木戸浩貴, 亀田倫史, 神谷昌克, 菊川峰志, 相沢智康, 藤原敏道, 出村誠 Abstr Annu Meet NMR Soc Jpn 53rd 298 -299 2014年11月04日 [査読無し][通常論文]
  • 冨澤聡, 神谷昌克, 菊川峰志, 出村誠, 相沢智康 日本蛋白質科学会年会プログラム・要旨集 14th 145 2014年05月26日 [査読無し][通常論文]
  • 田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠 Abstr Annu Meet NMR Soc Jpn 52nd 272 -273 2013年11月12日 [査読無し][通常論文]
  • 木戸浩貴, 田巻初, 江川文子, 亀田倫史, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠 Abstr Annu Meet NMR Soc Jpn 52nd 286 -287 2013年11月12日 [査読無し][通常論文]
  • TAKANASHI Yoshihiko, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, AIZAWA Tomoyasu, KAWANO Keiichi, DEMURA Makoto Peptide science : proceedings of the ... Japanese Peptide Symposium 2012 333 -336 2013年03月01日
  • SATO Yuji, TOMISAWA Satoshi, AIZAWA Tomoyasu, KAMIYA Masakatsu, KIKUKAWA Takashi, KUMAKI Yasuhiro, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2012 209 -212 2013年03月01日
  • TAKAHASHI Rika, KAMIYA Masakatsu, AIZAWA Tomoyasu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2012 51 -54 2013年03月01日
  • 出村 誠 キチン・キトサン研究 = Chitin and chitosan research 18 (3) 259 -261 2012年10月01日 [査読無し][通常論文]
  • 木崎皓太, 富張瑞樹, 大津航, 新敷信人, 塚本卓, 宮園耕介, 菊川峰志, 出村誠, 大塚弥生, 佐藤耕太, 高桑雄一, 稲葉睦 日本獣医学会学術集会講演要旨集 154th 338 2012年08月31日 [査読無し][通常論文]
  • 出村 誠 生物物理 52 (1) S7 2012年08月15日 [査読無し][通常論文]
  • 相沢 智康, 出村 誠, 河野 敬一 蚕糸・昆虫バイオテック = Sanshi-konchu biotec 81 (2) 115 -123 2012年08月01日 [査読無し][通常論文]
     
    現在地球上の陸上動物で最も繁栄しているグループは昆虫であり，昆虫は全動物種の80%以上を占めていると考えられている。多様な環境に適応してきた昆虫は，その生育域を広げるために，病原微生物から身を守る生体防御機構をはじめ，外敵から身を守り生き延びるための様々な仕組みを進化させてきたと考えられる。我々のグループでは，カイコをはじめとする昆虫等の無脊椎動物由来の，生体防御関連のタンパク質やペプチドの構造生物学的な解析を進めてきた。抗体による巧みな生体防御機構，いわゆる獲得免疫をもたない無脊椎動物が，外敵から身を守るメカニズムの解明は，学術的な視点からはもちろんのこと，種々の産業への応用を考える上でも興味深いテーマである。脊椎動物とは大きく異なる生体防御機構在もつ無脊椎動物においても，生体防御系が液性免疫と細胞性免疫からなるという点は脊椎動物と変わらない。液性免疫は体液中に存在する抗菌タンパク質やペプチドをはじめとする様々な生体防御分子による防御機構であり，細胞性免疫は血球細胞による異物の除去を中心とする防御機構である。本稿では，液性免疫を担う抗菌タンパク質の一種であるリゾチーム，および，細胞性免疫において重要な役割を果たすサイトカインであるENFペプチドファミリーについて，鱗翅目昆虫をターゲットにした我々のグループの研究成果を中心に紹介する。
  • 出村 誠 キチン・キトサン研究 = Chitin and chitosan research 18 (2) 87 -88 2012年07月01日 [査読無し][通常論文]
  • 原田 尚樹, 柏崎 晴彦, 赤澤 敏之, 村田 勝, 相沢 智康, 出村 誠, 田中 順三, 飯塚 正, 井上 農夫男 キチン・キトサン研究 = Chitin and chitosan research 18 (2) 112 -113 2012年07月01日 [査読無し][通常論文]
  • 櫛引 崇弘, 神谷 昌克, 相沢 智康, 境 勝義, 石川 和裕, 東 乙比古, 熊木 康裕, 菊川 峰志, 出村 誠, 川端 俊一郎, 河野 敬一 キチン・キトサン研究 = Chitin and chitosan research 18 (2) 184 -184 2012年07月01日 [査読無し][通常論文]
  • 山本愛弓, 塚本卓, 小橋川敬博, 内田毅, 出村誠, 稲垣冬彦, 石森浩一郎 日本蛋白質科学会年会プログラム・要旨集 12th 63 2012年05月31日 [査読無し][通常論文]
  • 菊川峰志, 小久保麻実, 塚本卓, 井原邦夫, 加茂直樹, 出村誠 日本蛋白質科学会年会プログラム・要旨集 12th 130 2012年05月31日 [査読無し][通常論文]
  • 木崎皓太, 富張瑞樹, 大塚弥生, 塚本卓, 新敷信人, 菊川峰志, 出村誠, 大津航, 佐藤耕太, 高桑雄一, 稲葉睦 日本膜学会年会講演要旨集 34th 63 2012年04月27日 [査読無し][通常論文]
  • 原田 尚樹, 柏崎 晴彦, 赤澤 敏之, 村田 勝, 相沢 智康, 出村 誠, 田中 順三, 飯塚 正, 井上 農夫男 北海道歯学雑誌 32 (2) 166 -176 2012年03月15日 [査読無し][通常論文]
     
    【背景・目的】ハイドロキシアパタイト（HAp）は生体親和性と骨伝導性に優れた生体材料であるが，その硬さと脆性のため，望む形状に成形することが困難である．それゆえ，HApの欠点である成形性を改善するHAp/高分子の新規複合材料の開発に多くの関心が集まっている．キトサンは甲殻類の外殻などに含まれる天然高分子で，その生体吸収性や高い熱安定性などの性質から生体材料として注目されている．我々はこれまでに，多孔性キトサン/HAp複合体を作製し，歯槽骨再生材料に適した柔軟性のある物性を持つことを報告してきた．本研究では，この複合体の骨形成蛋白質（rhBMP-2）担体としての有用性を評価する目的で，ラット頭頂骨骨膜下埋入実験を行い，骨形成過程と担体複合体の吸収変化を組織形態学的に検討した．【方法】共沈澱法とポローゲンリーチング法により多孔性キトサン/HAp複合体を作製し，５μg のrhBMP-2を添加した．rhBMP-2無添加の複合体を対照として，10週齢のSDラット頭頂骨骨膜下に埋入し，４，８週後に屠殺し，組織学的観察および形態計測を行った．【結果】rhBMP-2添加多孔性キトサン/HAp複合体では，埋入４，８週後に複合体の周辺部から中央部にかけて多数の細胞侵入および骨形成が認められた．対照群では骨形成はみられなかった．形態計測した結果，埋入４，８週後における複合体の占有率はrhBMP-2添加群では対照群に比べ有意に減少し（p＜0.05），骨形成に伴う複合体の吸収が認められた．【考察・結論】成形性や操作性の高い多孔性キトサン/HAp複合体は，rhBMP-2の担体として優れた骨形成能と生体吸収性を有することから，複雑な形態を呈する骨欠損部における生体材料として有用であることが示唆された．
  • KUSHIBIKI Takahiro, KAMIYA Masakatsu, AIZAWA Tomoyasu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWABATA Shunichiro, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 225 -226 2012年03月01日
  • KITAMURA Yuki, AIZAWA Tomoyasu, KAMIYA Masakastu, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 375 -376 2012年03月01日
  • TAKAHASHI Rika, AIZAWA Tomoyasu, KAMIYA Masakatsu, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 39 -42 2012年03月01日
  • NAKAZUMI Taichi, SAKAI Chihiro, MAEHANA Shiori, AIZAWA Tomoyasu, HOJO Eri, MATSUMOTO Takashi, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 219 -220 2012年03月01日
  • TAKANASHI Yoshihiko, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, AIZAWA Tomoyasu, KAWANO Keiichi, DEMURA Makoto Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 223 -224 2012年03月01日
  • 田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠 日本蛋白質科学会年会プログラム・要旨集 12th 2012年
  • 田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠 Abstracts. Annual Meeting of the NMR Society of Japan 51st 2012年
  • 相沢 智康, 出村 誠, 河野 敬一 蚕糸・昆虫バイオテック 81 (2) 2_115 -2_123 2012年 [査読無し][通常論文]
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Makoto Demura, Toshifumi Nara, Naoki Kamo JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 106 87 -94 2012年01月 [査読無し][通常論文]
     
    Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration-time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370. (C) 2011 Elsevier B.V. All rights reserved.
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Makoto Demura, Toshifumi Nara, Naoki Kamo JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 106 87 -94 2012年01月 [査読無し][通常論文]
     
    Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration-time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370. (C) 2011 Elsevier B.V. All rights reserved.
  • Yasutaka Yamashita, Takashi Kikukawa, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Seiji Miyauchi, Naoki Kamo, Makoto Demura BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1808 (12) 2905 -2912 2011年12月 [査読無し][通常論文]
     
    Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes. (C) 2011 Elsevier B.V. All rights reserved.
  • Tetsuo Asakura, Hirohito Nishi, Aya Nagano, Ai Yoshida, Yasumoto Nakazawa, Masakatsu Kamiya, Makoto Demura BIOMACROMOLECULES 12 (11) 3910 -3916 2011年11月 [査読無し][通常論文]
     
    It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF10, consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The H-1, N-15, and C-13 chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a C-13 solid-state NMR study was performed on a C-13 selectively labeled model peptide, AGSGAG[3-C-13]A(7)GSGAGAGSGGT[2-C-13]G(19)R[1-C-13]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the beta-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins.
  • Tetsuo Asakura, Hirohito Nishi, Aya Nagano, Ai Yoshida, Yasumoto Nakazawa, Masakatsu Kamiya, Makoto Demura BIOMACROMOLECULES 12 (11) 3910 -3916 2011年11月 [査読無し][通常論文]
     
    It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF10, consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The H-1, N-15, and C-13 chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a C-13 solid-state NMR study was performed on a C-13 selectively labeled model peptide, AGSGAG[3-C-13]A(7)GSGAGAGSGGT[2-C-13]G(19)R[1-C-13]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the beta-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins.
  • Gang Dai, Yu Zhang, Jun Tamogami, Makoto Demura, Naoki Kamo, Hideki Kandori, Tatsuo Iwasa BIOCHEMISTRY 50 (33) 7177 -7183 2011年08月 [査読無し][通常論文]
     
    Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (lambda(max)) at 482 nm and can thermally revert back to spR above -160 degrees C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9=C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.
  • Gang Dai, Yu Zhang, Jun Tamogami, Makoto Demura, Naoki Kamo, Hideki Kandori, Tatsuo Iwasa BIOCHEMISTRY 50 (33) 7177 -7183 2011年08月 [査読無し][通常論文]
     
    Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (lambda(max)) at 482 nm and can thermally revert back to spR above -160 degrees C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9=C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.
  • KAMIYA Masakatsu, YOSHIDA Tomoshi, AIZAWA Tomoyasu, KIKUKAWA Takashi, IMAI Kunio, SUZUKI Koichi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 160 -160 2011年03月01日
  • SASAKI Takanori, DEMURA Makoto, KATO Noritaka, MUKAI Yuri Biochemistry 50 (12) 2283 -2290 2011年03月 [査読無し][通常論文]
     
    A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl beta-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR
  • Takanori Sasaki, Makoto Demura, Noritaka Kato, Yuri Mukai BIOCHEMISTRY 50 (12) 2283 -2290 2011年03月 [査読無し][通常論文]
     
    A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl beta-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR
  • 田巻初, 江川文子, 松木陽, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠 日本蛋白質科学会年会プログラム・要旨集 11th 2011年
  • Demura Makoto 高等教育ジャ-ナル (18) 63 -71 2011年01月 [査読無し][通常論文]
  • Kazunari Higa, Naomi Takeshima, Fumika Moro, Tetsuya Kawakita, Motoko Kawashima, Makoto Demura, Jun Shimazaki, Tetsuo Asakura, Kazuo Tsubota, Shigeto Shimmura JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 22 (17) 2261 -2276 2011年 [査読無し][通常論文]
     
    Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin beta 1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium. (C) Koninklijke Brill NV, Leiden, 2011
  • Takahide Kouno, Nobuhisa Watanabe, Naoki Sakai, Takashi Nakamura, Yuko Nabeshima, Masashi Morita, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Tsuneo Imanaka, Isao Tanaka, Keiichi Kawano JOURNAL OF MOLECULAR BIOLOGY 405 (2) 560 -569 2011年01月 [査読無し][通常論文]
     
    Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a beta-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the beta-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down beta-sheets. This up-and-down beta-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down beta-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down beta-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain. (C) 2010 Elsevier Ltd. All rights reserved.
  • Biochim. Biophys. Acta 1808 2905 -2912 2011年 [査読無し][通常論文]
  • Kazunari Higa, Naomi Takeshima, Fumika Moro, Tetsuya Kawakita, Motoko Kawashima, Makoto Demura, Jun Shimazaki, Tetsuo Asakura, Kazuo Tsubota, Shigeto Shimmura JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 22 (17) 2261 -2276 2011年 [査読無し][通常論文]
     
    Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin beta 1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium. (C) Koninklijke Brill NV, Leiden, 2011
  • Wei Wang, Soichiro Itoh, Tomoyasu Aizawa, Atsushi Okawa, Katsuyoshi Sakai, Tsuneo Ohkuma, Makoto Demura BIOMEDICAL MATERIALS 5 (6) 065009 2010年12月 [査読無し][通常論文]
     
    A chitosan/marine-originated collagen composite has been developed. This composite gel was characterized and its biocompatibility, as well as an inflammatory reaction, was observed. The chitosan gel including N-3-carboxypropanoil-6-O-(carboxymethyl) chitosan of 3 mol%, 6-O-(carboxymethyl) chitosan of 62 mol% and 6-O-(carboxymethyl) chitin of 35 mol% was prepared and compounded with the salmon atelocollagen (SA) gel at different mixture ratios. The composite gels were injected subcutaneously in to the back of rats. The specimens were harvested for a histological survey as well as a tumor necrosis factor-alpha (TNF-alpha) assay by ELISA. The inflammatory cell infiltration and release of TNF-alpha were successively controlled low with the ratio of SA to chitosan at 10:90 or 20:80. The SA gel first, within 2 weeks, and then chitosan in the composite gel were slowly absorbed after implantation, followed by soft tissue formation. It is expected that this composite gel will be available as a carrier for tissue filler and drug delivery systems.
  • Wei Wang, Soichiro Itoh, Tomoyasu Aizawa, Atsushi Okawa, Katsuyoshi Sakai, Tsuneo Ohkuma, Makoto Demura BIOMEDICAL MATERIALS 5 (6) 065009 2010年12月 [査読無し][通常論文]
     
    A chitosan/marine-originated collagen composite has been developed. This composite gel was characterized and its biocompatibility, as well as an inflammatory reaction, was observed. The chitosan gel including N-3-carboxypropanoil-6-O-(carboxymethyl) chitosan of 3 mol%, 6-O-(carboxymethyl) chitosan of 62 mol% and 6-O-(carboxymethyl) chitin of 35 mol% was prepared and compounded with the salmon atelocollagen (SA) gel at different mixture ratios. The composite gels were injected subcutaneously in to the back of rats. The specimens were harvested for a histological survey as well as a tumor necrosis factor-alpha (TNF-alpha) assay by ELISA. The inflammatory cell infiltration and release of TNF-alpha were successively controlled low with the ratio of SA to chitosan at 10:90 or 20:80. The SA gel first, within 2 weeks, and then chitosan in the composite gel were slowly absorbed after implantation, followed by soft tissue formation. It is expected that this composite gel will be available as a carrier for tissue filler and drug delivery systems.
  • Yasuhiro Nonaka, Hideki Muto, Tomoyasu Aizawa, Etsuro Okabe, Shohei Myoba, Takuya Yokoyama, Shin Saito, Fumie Tatami, Yasuhiro Kumaki, Masakatsu Kamiya, Takashi Kikukawa, Mineyuki Mizuguchi, Shigeharu Takiya, Masataka Kinjo, Makoto Demura, Keiichi Kawano BIOCHEMISTRY 49 (38) 8367 -8375 2010年09月 [査読無し][通常論文]
     
    The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and a-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in a-helix structure content.
  • Masaki Takahashi, Mineyuki Mizuguchi, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Hitoshi Okazawa, Keiichi Kawano BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1804 (7) 1500 -1507 2010年07月 [査読無し][通常論文]
     
    Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts with various proteins, including RNA polymerase II and the spliceosomal protein U5-15kD. PQBP-1 is known to be associated with X-linked mental retardation in which a frameshift mutation in the PQBP-1 gene occurs. In the present study, we demonstrate that PQBP-1 binds to U5-15kD via a continuous 23-residue segment within its C-terminal domain. Intriguingly, this segment is lost in the frameshift mutants of PQBP-1 associated with X-linked mental retardation. These findings suggest that the frameshift mutations in the PQBP-1 gene lead to expression of mutants lacking the ability to interact with U5-15kD. (C) 2010 Elsevier B.V. All rights reserved.
  • T. Sasaki, M. Demura, Y. Mukai FEBS JOURNAL 277 243 -243 2010年06月 [査読無し][通常論文]
  • Masakatsu Kamiya, Keisuke Oyauchi, Yoshinori Sato, Takuya Yokoyama, Mofei Wang, Tomoyasu Aizawa, Yasuhiro Kumaki, Mineyuki Mizuguchi, Kunio Imai, Makoto Demura, Koichi Suzuki, Keiichi Kawano JOURNAL OF PEPTIDE SCIENCE 16 (5) 242 -248 2010年05月 [査読無し][通常論文]
     
    We previously reported that yamamarin, a pentapeptide with an amidated C-terminus (DILRG-NH(2)) isolated from larvae of the silkmoth, and its palmitoylated analog (C16-DILRG-NH(2)) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure-activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C-terminal part (-RG-NH(2)) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG-NH(2) and C16-DILRG-NH(2) revealed that the N-terminal palmitoyl group of C16-DILRG-NH(2) did not affect the conformation of the C-terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright (C) 2010 European Peptide Society and John Wiley & Sons, Ltd.
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Ayaka Takemura, Makoto Demura, Naoki Kamo BIOPHYSICAL JOURNAL 98 (7) 1353 -1363 2010年04月 [査読無し][通常論文]
     
    Sensory rhodopsin II (HsSRII, also called phoborhodopsin) is a negative phototaxis receptor of Halobacterium salinarum, a bacterium that avoids blue-green light. In this study, we expressed the protein in Escherichia colt cells, and reconstituted the purified protein with phosphatidylcholine. The reconstituted HsSRII was stable. We examined the photocycle by flash-photolysis spectroscopy in the time range of milliseconds to seconds, and measured proton uptake/release using a transparent indium-tin oxide electrode. The pKa of the counterion of the Schiff base, Asp(73), was 3.0. Below pH 3, the depleted band was observed on flash illumination, but the positive band in the difference spectra was not found. Above pH 3, the basic photocycle was HsSRII (490) -> M (350) -> O (520) -> Y (490) -> HsSRII, where the numbers in parentheses are the maximum wavelengths. The decay rate of O-intermediate and Y-intermediate were pH-independent, whereas the M-intermediate decay was pH-dependent. For 3 < pH < 4.5, the M-decay was one phase, and the rate decreased with an increase in pH. For 4.5 < pH < 6.5, the decay was one phase with pH-independent rates, and azide markedly accelerated the M-decay. These findings suggest the existence of a protonated amino acid residue (X-H) that may serve as a proton relay to reprotonate the Schiff base. Above pH 6.5, the M-decay showed two phases. The fast M-decay was pH-independent and originated from the molecule having a protonated X-H, and the slow M-decay originated from the molecule having a deprotonated X, in which the proton came directly from the outside. The analysis yielded a value of 7.5 for the pKa of X-H. The proton uptake and release occurred during M-decay and O-decay, respectively.
  • Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Ayaka Takemura, Makoto Demura, Naoki Kamo BIOPHYSICAL JOURNAL 98 (7) 1353 -1363 2010年04月 [査読無し][通常論文]
     
    Sensory rhodopsin II (HsSRII, also called phoborhodopsin) is a negative phototaxis receptor of Halobacterium salinarum, a bacterium that avoids blue-green light. In this study, we expressed the protein in Escherichia colt cells, and reconstituted the purified protein with phosphatidylcholine. The reconstituted HsSRII was stable. We examined the photocycle by flash-photolysis spectroscopy in the time range of milliseconds to seconds, and measured proton uptake/release using a transparent indium-tin oxide electrode. The pKa of the counterion of the Schiff base, Asp(73), was 3.0. Below pH 3, the depleted band was observed on flash illumination, but the positive band in the difference spectra was not found. Above pH 3, the basic photocycle was HsSRII (490) -> M (350) -> O (520) -> Y (490) -> HsSRII, where the numbers in parentheses are the maximum wavelengths. The decay rate of O-intermediate and Y-intermediate were pH-independent, whereas the M-intermediate decay was pH-dependent. For 3 < pH < 4.5, the M-decay was one phase, and the rate decreased with an increase in pH. For 4.5 < pH < 6.5, the decay was one phase with pH-independent rates, and azide markedly accelerated the M-decay. These findings suggest the existence of a protonated amino acid residue (X-H) that may serve as a proton relay to reprotonate the Schiff base. Above pH 6.5, the M-decay showed two phases. The fast M-decay was pH-independent and originated from the molecule having a protonated X-H, and the slow M-decay originated from the molecule having a deprotonated X, in which the proton came directly from the outside. The analysis yielded a value of 7.5 for the pKa of X-H. The proton uptake and release occurred during M-decay and O-decay, respectively.
  • 田巻初, 樋口真理花, 江川文子, 藤原敏道, 横山順, 横山順, 横山順, 木川隆則, 木川隆則, 下野和実, 下野和実, 染谷友美, 白水美香子, 横山茂之, 横山茂之, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 出村誠 日本蛋白質科学会年会プログラム・要旨集 10th 2010年
  • 田巻初, 樋口真理花, 江川文子, 藤原敏道, 横山順, 横山順, 横山順, 木川隆則, 木川隆則, 下野和実, 下野和実, 染谷友美, 白水美香子, 横山茂之, 横山茂之, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 出村誠 Abstracts. Annual Meeting of the NMR Society of Japan 49th 2010年
  • 出村 誠 生物物理 49 (6) 279 -279 2009年12月25日 [査読無し][通常論文]
  • Masashi Sonoyama, Takashi Kikukawa, Yasunori Yokoyama, Makoto Demura, Naoki Kamo, Shigeki Mitaku CHEMISTRY LETTERS 38 (12) 1134 -1135 2009年12月 [査読無し][通常論文]
     
    The photocycle of bacteriorhodopsin (bR) reconstituted into dimyristoylphosphatidylcholine vesicles was investigated with transient visible absorption spectroscopy. The measured time-resolved difference spectra indicated that two substates of the M intermediate with almost the same absorption maximum were observed in the gel state, whereas the spectrum of M showed a splitting into an early and a late component shifted by approximately 15 nm in the liquid crystalline phase, suggesting disassembly of bR molecules induces remarkable structural changes around the retinal pocket of the late M intermediate responsible for switching protein conformation from a proton release form to a proton uptake form.
  • 塚本卓, LI Xiang Lan, 花方信孝, 出村誠 Abstr Annu Meet NMR Soc Jpn 48th 166 -167 2009年11月10日 [査読無し][通常論文]
  • Yoshitaka Umetsu, Tomoyasu Aizawa, Kaori Muto, Hiroko Yamamoto, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano JOURNAL OF BIOLOGICAL CHEMISTRY 284 (43) 29625 -29634 2009年10月 [査読無し][通常論文]
     
    Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.
  • 河野隆英, 藤谷直樹, 水口峰之, 大崎司, 川畑俊一郎, 相沢智康, 出村誠, 河野敬一 生化学 ROMBUNNO.2P-443 2009年09月25日 [査読無し][通常論文]
  • Takahide Kouno, Mineyuki Mizuguchi, Tomoyasu Aizawa, Hiroyuki Shinoda, Makoto Demura, Shun-ichiro Kawabata, Keiichi Kawano BIOCHEMISTRY 48 (32) 7629 -7635 2009年08月 [査読無し][通常論文]
     
    Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. The amino acid sequence of big defensin is divided into an N-terminal hydrophobic domain and a C-terminal cationic domain, Which are responsible for antimicrobial activities against Gram-positive and -negative bacteria, respectively. The N-terminal domain of big defensin forms a unique globular conformation with two alpha-helices and a parallel beta-sheet, while the C-terminal domain adopts a beta-defensin-like fold. Although Our previous study implied that big defensin changes its N-terminal structure in a micellar environment, due to the poor quality of the NMR spectra it remained to be resolved whether the N-terminal domain adopts any Structure in the presence of micelles. In this analysis, We Successfully determined the structure of the N-terminal fragment of big defensin in a micellar solution, showing that the fragment peptide forms a single a-helix structure. Moreover, NMR experiments using paramagnetic probes revealed that the N-terminal domain of big defensin penetrates into the micelle with a dipping at the N-terminal edge of the alpha-helix. Here, we propose a model for how big defensin associates with the target membrane.
  • Shigeharu Takiya, Shin Saito, Takuya Yokoyama, Daisuke Matsumoto, Tomoyasu Aizawa, Masakatsu Kamiya, Makoto Demura, Keiichi Kawano JOURNAL OF BIOCHEMISTRY 146 (1) 103 -111 2009年07月 [査読無し][通常論文]
     
    The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino-acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.
  • Masaki Takahashi, Mineyuki Mizuguchi, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Hitoshi Okazawa, Keiichi Kawano BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1794 (6) 936 -943 2009年06月 [査読無し][通常論文]
     
    Polyglutamine tract binding protein-1 (PQBP-1) is a nuclear protein that interacts with disease proteins containing expanded polyglutamine repeats. PQBP-1 also interacts with RNA polymerase II and a spliceosomal protein U5-15kD. In the present study, we demonstrate that PQBP-1 is composed of a large unstructured region and a small folded core. Intriguingly, the large unstructured region encompasses two functional domains: a polar amino acid rich domain and a C-terminal domain. These findings suggest that PQBP-1 belongs to the family of intrinsically unstructured/disordered proteins. Furthermore, the binding of the target molecule U5-15kD induces only minor conformational changes into PQBP-1. Our results suggest that PQBP-1 includes high content of unstructured regions in the C-terminal domain, in spite of the binding of U5-15kD. (C) 2009 Elsevier B.V. All rights reserved.
  • Keisuke Nakashima, Takumi Nakamura, Satoshi Takeuchi, Mikihiro Shibata, Makoto Demura, Tahei Tahara, Hideki Kandori JOURNAL OF PHYSICAL CHEMISTRY B 113 (24) 8429 -8434 2009年06月 [査読無し][通常論文]
     
    Halorhodopsin (HR) is a light-driven chloride pump. Cl- is bound in the Schiff base region of the retinal chromophore, and unidirectional Cl- transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. It is known that HR from Natronobacterium pharaonis (pHR) also pumps NO3- with similar efficiency, suggesting that NO3- binds to the Cl--binding site. In the present study, we investigated the properties of the anion-binding site by means of ultrafast pump-probe spectroscopy and low-temperature FTIR spectroscopy. The obtained data were surprisingly similar between pHR-NO3- and pHR-Cl-, even though the shapes and sizes of the two anions are quite different. Femtosecond pump-probe spectroscopy showed very similar excited-state dynamics between pHR-NO3- and pHR-Cl-. Low-temperature FTIR spectroscopy of unlabeled and [zeta-N-15]Lys-labeled pHR revealed almost identical hydrogen-bonding strengths of the protonated retinal Schiff base between pHR-NO3- and pHR-Cl-, which is similarly strengthened after retinal isomerization. There were spectral variations for water stretching vibrations between pHR-NO3- and pHR-Cl-, suggesting that the water molecules hydrate each anion. Nevertheless, the overall spectral features were similar for the two species. These observations strongly suggest that the anion-binding site has a flexible structure and that the interaction between retinal and the anions is weak, despite the presence of an electrostatic interaction. Such a flexible hydrogen-bonding network in the Schiff base region in HR appears to be in remarkable contrast to that in light-driven proton-pumping proteins.
  • Shin-ichi Nakatogawa, Yasunori Oda, Masakatsu Kamiya, Tatsuro Kamijima, Tomoyasu Aizawa, Kevin D. Clark, Makoto Demura, Keiichi Kawano, Michael R. Strand, Yoichi Hayakawa CURRENT BIOLOGY 19 (9) 779 -785 2009年05月 [査読無し][通常論文]
     
    Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.
  • Keiichi Inoue, Megumi Kubo, Makoto Demura, Naoki Kamo, Masahide Terazima BIOPHYSICAL JOURNAL 96 (9) 3724 -3734 2009年05月 [査読無し][通常論文]
     
    Reaction dynamics of a chloride ion pump protein, halorhodopsin (HR), from Natronomonas pharaonis (N. pharaonis) (NpHR) was studied by the pulsed-laser-induced transient grating (TG) method. A detailed investigation of the TG signal revealed that there is a spectrally silent diffusion process besides the absorption-observable reaction dynamics. We interpreted these dynamics in terms of release, diffusion, and uptake of the Cl- ion. From a quantitative global analysis of the signals at various grating wavenumbers, it was concluded that the release of the Cl- ion is associated with the L2 -> (L2 (or N) reversible arrow O) process, and uptake of Cl- occurs with the (L2 (or N) reversible arrow O) -> NpHR' process. The diffusion coefficient of NpHR solubilized in a detergent did not change during the cyclic reaction. This result contrasts the behavior of many photosensor proteins and implies that the change in the H-bond network from intra- to intermolecular is not significant for the activity of this protein pump.
  • Yasuhiro Nonaka, Daisuke Akieda, Tomoyasu Aizawa, Nobuhisa Watanabe, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Takashi Kikukawa, Makoto Demura, Keiichi Kawano FEBS JOURNAL 276 (8) 2192 -2200 2009年04月 [査読無し][通常論文]
     
    In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.
  • OYAUCHI Keisuke, KAMIYA Masakatsu, YOKOYAMA Takuya, WANG Mofei, AIZAWA Tomoyasu, DEMURA Makoto, SUZUKI Koichi, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2008 269 -270 2009年03月01日
  • Megumi Kubo, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 (2) 547 -555 2009年03月 [査読無し][通常論文]
     
    Halorhodopsin (HR) acts as a light-driven chloride pump which transports a chloride ion from the extracellular (EC) to the cytoplasmic space during a photocycle reaction that includes some photointermediates initiated by illumination. To understand the chloride uptake mechanisms, we focused on a basic residue Arg123 of HR from Natronomonas pharaonis (NpHR), which is the only basic residue located in the EC half ion channel. By the measurements of the visible absorption spectra in the dark and the light-induced inward current through the membrane, it was shown that the chloride binding and transport ability of NpHR completely disappeared by the change of arginine to glutamine. From flashphotolysis analysis, the photocycle of R123Q differed from that of wildtype NpHR completely. The response of the R123H mutant depended on pH. These facts imply that the positive charge at position 123 is essential for chloride binding in the ground state and for the chloride uptake under illumination. On the basis of the molecular structures of HR and the anion-transportable mutants of bacteriorhodopsin, the effects of the positive charge and the conformational change of the Arg123 side chain as well as the chloride-pumping mechanism are discussed.
  • 樋口真理花, 江川文子, 田巻初, 神谷昌克, 相沢智康, 河野敬一, 藤原敏道, 出村誠 Abstracts. Annual Meeting of the NMR Society of Japan 48th 2009年
  • Mineyuki Mizuguchi, Tomoyasu Aizawa, Keiichi Kawano, Makoto Demura ANNUAL REPORTS ON NMR SPECTROSCOPY, VOL 65 65 53 -76 2009年 [査読有り][招待有り]
     
    NMR is a powerful tool for characterizing the structure and dynamics of proteins at the site-specific level. A variety of NMR techniques can be applied to study various conformational states during the folding process, including the highly unfolded, partially folded, and native states. One particularly powerful method is the combination of NMR with hydrogen-deuterium exchange to indicate the formation of hydrogen bonds in proteins. Examples of the application of a variety of NMR methods to the protein folding studies of alpha-lactalbumin and c-type lysozyme are presented.
  • Biochemistry in press 2009年 [査読無し][通常論文]
  • Biophys. J accepted 2009年 [査読無し][通常論文]
  • Takanori Sasaki, Megumi Kubo, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 (1) 130 -136 2009年01月 [査読無し][通常論文]
     
    Halorhodopsin (HR) is a transmembrane seven-helix retinal protein, and acts as an inward light-driven Cl(-) pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 mm) to 1% (20 mm) dodecyl-beta-d-maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 mm). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton-coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR-DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross-linked NpHR by glutaraldehyde gave the SDS-PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR (Escherichia coli and Halobacterium salinarum), CD spectra showed exciton-coupling, which suggests strongly the trimer structure in the cell membrane.
  • Hiroki Ishihara, Goh Matsuo, Takanori Sasaki, Yuko Saito, Makoto Demura, Kaoru Tsujii GELS: STRUCTURES, PROPERTIES, AND FUNCTIONS 136 143 -+ 2009年 [査読無し][通常論文]
     
    Electrophoresis of dye stuffs and proteins in poly (acrylamide) gel containing immobilized bilayer membranes have been studied. Bilayer membranes of a polymerizable surfactant, dodecylglyceryl itaconate (DGI), can be immobilized in poly(acrylamide) gels, and the hybrid gels are first applied to a substrate of the poly(acrylamide) gel electrophoresis (PAGE). The bilayer-membranes-immobilized-gel (abbreviated as BM-gel) showed different separation behaviors from those by the conventional PAGE. The separation behavior of dye stuffs suggests that the bilayer membranes in the BM-gel work as a separator of the test molecules due to their hydrophilic/hydrophobic nature. Water-soluble proteins migrated faster in the BM-gels than in the simple poly(acrylamide) gels. Membrane proteins, on the other hand, did not move at all in the BM-gels probably because the protein molecules were entrapped firmly inside the bilayer membranes.
  • Tatsuro Kamijima, Ayaka Ohmura, Toshiya Sato, Kaoru Akimoto, Miki Itabashi, Mineyuki Mizuguchi, Masakatsu Kamiya, Takashi Kikukawa, Tomoyasu Aizawa, Masayuki Takahashi, Keiichi Kawano, Makoto Demura BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 376 (1) 211 -214 2008年11月 [査読無し][通常論文]
     
    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, Circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes. (C) 2008 Elsevier Inc. All rights reserved.
  • Takumi Nakamura, Satoshi Takeuchi, Mikihiro Shibata, Makoto Demura, Hideki Kandori, Tahei Tahara JOURNAL OF PHYSICAL CHEMISTRY B 112 (40) 12795 -12800 2008年10月 [査読無し][通常論文]
     
    Halorhodopsin is a retinal protein that acts as a light-driven chloride pump in the Haloarchaeal cell membrane. A chloride ion is bound near the retinal chromophore, and light-induced all-trans -> 13-cis isomerization triggers the unidirectional chloride ion pump. We investigated the primary ultrafast dynamics of Natronomonas pharaonis halorhodopsin that contains Cl-, Br-, or I- (pHR-Cl-, pHR-Br-, or pHR-I-) using ultrafast pump-probe spectroscopy with similar to 30 fs time resolution. All of the temporal behaviors of the S-n <- S, absorption, ground-state bleaching, K intermediate (13-cis form) absorption, and stimulated emission were observed. In agreement with previous reports, the primary process exhibited three dynamics. The first dynamics corresponds to the population branching process from the Franck-Condon (FC) region to the reactive (S-1(r)) and non-reactive (S-1(nr)) S, states. With the improved time resolution, it was revealed that the time constant of this branching process (tau(1)) is as short as 50 fs. The second dynamics was the isomerization process of the S-1(r) state to generate the ground-state 13-cis form, and the time constant (tau(2)) exhibited significant halide ion dependence (1.4, 1.6, and 2.2 ps for pHR-Cl-, pHR-Br-, and pHR-I-, respectively). The relative quantum yield of the isomerization, which was evaluated from the pump-probe signal after 20 ps, also showed halide ion dependence (1.00, 1. 14, and 1.35 for pHR-Cl-, pHR-Br-, and pHR-I-, respectively). It was revealed that the halide ion that accelerates isomerization dynamics provides the lower isomerization yield. This finding suggests that there is an activation barrier along the isomerization coordinate on the S, potential energy surface, meaning that the three-state model, which is now accepted for bacteriorhodopsin, is more relevant than the two-state model for the isomerization process of halorhodopsin. We concluded that, with the three-state model, the isomerization rate is controlled by the height of the activation barrier on the S, potential energy surface while the overall isomerization yield is determined by the branching ratios at the FC region and the conical intersection. The third dynamics attributable to the internal conversion of the S-1(nr) state also showed notable halide ion dependence (tau(3) = 4.5, 4.6, and 6.3 ps for pHR-Cl-, pHR-Br-, and pHR-I-). This suggests that some geometrical change may be involved in the relaxation process of the S-1(nr) state.
  • Takahide Kouno, Naoki Fujitani, Mineyuki Mizuguchi, Tsukasa Osaki, Shin-ichiro Nishimura, Shun-ichiro Kawabata, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta, Keiichi Kawan BIOCHEMISTRY 47 (40) 10611 -10619 2008年10月 [査読無し][通常論文]
     
    Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a P-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.
  • Shin Saito, Takuya Yokoyama, Tomoyasu Aizawa, Kyosuke Kawaguchi, Takeshi Yamaki, Daisuke Matsumoto, Tatsuro Kamijima, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Sigeharu Takiya, Makoto Demura, Keiichi Kawano PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 (1) 414 -426 2008年07月 [査読無し][通常論文]
     
    Fibroin-modulator-binding protein 1 (FMBP-1) is a predicted transcription factor of the silkworm fibroin gene. The DNA-binding domain of FMBP-1 consists of four almost perfect tandem repeats of 23 amino acids each (R1-R4), and is referred to as the score and three amino acid peptide repeat (STPR) domain. This characteristic domain is conserved in eukaryotes, but the DNA-binding mode is not known. In this study, the structural properties of the DNA-bound form of the STPR domain were characterized. The combined experiments indicated that the STPR domain bound to the DNA duplex wit a 1:1 binding ratio. The specific DNA caused considerable changes in the thermal unfolding profile and the digestion pattern of the STPR domain. These data suggested that the domain adapts a quite rigid helix-rich structure in the DNA-bound state, even though it moves flexibly in the absence of DNA. Furthermore, mutual induced-fit conformational change was also observed in DNA. Finally, we determined the DNA-binding surface of the STPR third repeat (R3) by alamne scanning mutagenesis; a particular site, composed of hydrophobic and hydrophilic residues, was identified. Notably, the substitution of Arg-9 in R3 with alanine residue, which is located in the middle of the surface, drastically abolished the -i-helix-inducing and DNA-binding abilities. From these results, we predicted the DNA-binding mode of the STPR domain.
  • Yasuhiro Nonaka, Tomoyasu Aizawa, Daisuke Akieda, Masanori Yasui, Masahiro Watanabe, Nobuhisa Watanabe, Isao Tanaka, Masakatsu Kamiya, Mineyuki Mizuguchi, Makoto Demura, Keiichi Kawano PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 (1) 313 -322 2008年07月 [査読無し][通常論文]
     
    Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.
  • Mineyuki Mizuguchi, Ayumi Hayashi, Makoto Takeuchi, Mizuki Dobashi, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 (1) 261 -269 2008年07月 [査読無し][通常論文]
     
    Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wildtype transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C-terminal-fragments starting near amino acid residue 50 as well as full-length transthyretin. Although a number of previous studies suggest the importance of the C-terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C-terminal fragments of transthyretin. To understand the role of C-terminal fragments in SSA, we examined the effects of the truncation of the N-terminal portions on the structure and aggregation properties of wild-type transthyretin. The deletion mutant lacking 50 N-terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self-assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N-terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self-assembly into aggregations in SSA.
  • T. Kouno, M. Mizuguchi, H. Tanaka, P. Yang, Y. Mori, H. Shinoda, K. Unoki, T. Aizawa, M. Demura, K. Suzuki, K. Kawano FEBS JOURNAL 275 167 -167 2008年06月 [査読無し][通常論文]
  • Wei Wang, Soichiro Itoh, Atsushi Matsuda, Tomoyasu Aizawa, Makoto Demura, Shizuko Ichinose, Kenichi Shinomiya, Junzo Tanaka JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 85A (4) 919 -928 2008年06月 [査読無し][通常論文]
     
    We have developed a novel bilayered chitosan tube that comprises an outer layer of chitosan film and an inner layer of chitosan nonwoven nano/microfiber mesh. The tube is fabricated with an electrospinning method. We characterized the microstructure and mechanical properties of this material. We introduced glycine spacers into the CYIGSR sequence, domain of laminin-1 that enhances Schwann cells migration and attachment, as well as neural outgrowth, resulting in the amino acid sequences CGGYIGSR and CGGGGGGYIGSR. These peptides were covalently bound to the nano/microfiber mesh surface of the chitosan tube to examine the effects of peptide mobility on nerve regeneration. Scaffolds constructed from these bilayered chitosan tubes were grafted to bridge injured sciatic nerve. Isografting was performed as a control. These scaffolds were removed 5 and 10 weeks after implantation for histological analysis. Nerve regeneration into chitosan tubes, on which the CGGGGGGYIGSR peptide was immobilized, exhibited efficacy similar to that of the isograft and represent a promising candidate for promoting peripheral nerve repair. (C) 2007 Wiley Periodicals, Inc.
  • UMETSU Yoshitaka, AIZAWA Tomoyasu, MUTO Kaori, YAMAMOTO Hiroko, KAMIYA Masakatsu, KUMAKI Yasuhiro, MIZUGUCHI Mineyuki, DEMURA Makoto, HAYAKAWA Yoichi, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 251 -254 2008年03月01日
  • SUGITA Keitaro, KAMIYA Masakatsu, OHKUBO Tomoyuki, KAMIJIMA Tatsuro, SHIMAMOTO Satoshi, TATAMI Fumie, AIZAWA Tomoyasu, MIZUGUCHI Mineyuki, KAWABATA Shun-ichiro, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 247 -248 2008年03月01日
  • KOUNO Takahide, MIZUGUCHI Mineyuki, TANAKA Hiromasa, YANG Ping, AIZAWA Tomoyasu, DEMURA Makoto, SUZUKI Koichi, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 75 -78 2008年03月01日
  • 樋口真理花, 江川文子, 神谷昌克, 相沢智康, 河野敬一, 藤原敏道, 出村誠 Abstracts. Annual Meeting of the NMR Society of Japan 47th 2008年
  • Takahide Kouno, Mineyuki Mizuguchi, Hiromasa Tanaka, Ping Yang, Yoshihiro Mori, Hiroyuki Shinoda, Kana Unoki, Tomoyasu Aizawa, Makoto Demura, Koichi Suzuki, Keiichi Kawano BIOCHEMISTRY 46 (48) 13733 -13741 2007年12月 [査読無し][通常論文]
     
    Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ Channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional H-1 nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungai peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.
  • Takahide Kouno, Mineyuki Mizuguchi, Hiromasa Tanaka, Ping Yang, Yoshihiro Mori, Hiroyuki Shinoda, Kana Unoki, Tomoyasu Aizawa, Makoto Demura, Koichi Suzuki, Keiichi Kawano BIOCHEMISTRY 46 (48) 13733 -13741 2007年12月 [査読無し][通常論文]
     
    Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ Channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional H-1 nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungai peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.
  • 塚本 卓, 佐々木 貴規, 久保 恵美, 神谷 昌克, 相沢 智康, 河野 敬一, 出村 誠 生物物理 47 (1) S46 2007年11月20日 [査読無し][通常論文]
  • 杉田 圭太郎, 神谷 昌克, 大久保 知行, 上島 達郎, 島本 怜史, 多々見 文恵, 相沢 智康, 水口 峰之, 川畑 俊一郎, 出村 誠, 河野 敬一 生物物理 47 (1) S125 2007年11月20日 [査読無し][通常論文]
  • 相沢 智康, 齊藤 伸, 横山 卓也, 上島 達朗, 神谷 昌克, 熊木 康裕, 滝谷 重治, 出村 誠, 河野 敬一 生物物理 47 (1) S125 2007年11月20日 [査読無し][通常論文]
  • 野中 康宏, 秋枝 大介, 神谷 昌克, 相沢 智康, 出村 誠, 河野 敬一 生物物理 47 (1) S130 2007年11月20日 [査読無し][通常論文]
  • 宮内 正二, 関 顕照, 菊川 峰志, 青山 健太郎, 出村 誠, Ganapathy Vadivel, 加茂 直樹 生物物理 47 (1) S188 2007年11月20日 [査読無し][通常論文]
  • 久保 恵美, 菊川 峰志, 宮内 正二, 関 顕照, 神谷 昌克, 相沢 智康, 河野 敬一, 加茂 直樹, 出村 誠 生物物理 47 (1) S258 2007年11月20日 [査読無し][通常論文]
  • 菊川 峰志, 重村 洋明, 久保 恵美, 出村 誠, 宮内 正二, 加茂 直樹 生物物理 47 (1) S258 2007年11月20日 [査読無し][通常論文]
  • 中島 啓介, 柴田 幹大, 出村 誠, 神取 秀樹 生物物理 47 (1) S259 2007年11月20日 [査読無し][通常論文]
  • 柴田 幹大, 出村 誠, 神取 秀樹 生物物理 47 (1) S259 2007年11月20日 [査読無し][通常論文]
  • 中村 巧, 竹内 佐年, 柴田 幹大, 出村 誠, 神取 秀樹, 田原 太平 生物物理 47 (1) S259 2007年11月20日 [査読無し][通常論文]
  • 樋口 真理花, 江川 文子, 神谷 昌克, 相沢 智康, 河野 敬一, 藤原 敏道, 出村 誠 生物物理 47 (1) S259 2007年11月20日 [査読無し][通常論文]
  • Hiroyasu Nakatani, Kosuke Maki, Kimiko Saeki, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Shuji Tomoda, Kunihiro Kuwajima BIOCHEMISTRY 46 (17) 5238 -5251 2007年05月 [査読無し][通常論文]
     
    The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The Phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.
  • Naoki Fujitani, Takahide Kouno, Taku Nakahara, Kenji Takaya, Tsukasa Osaki, Shun-Ichiro Kawabata, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Shin-Ichiro Nishimura, Keiichi Kawano JOURNAL OF PEPTIDE SCIENCE 13 (4) 269 -279 2007年04月 [査読無し][通常論文]
     
    Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 angstrom, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.
  • Akiteru Seki, Seiji Miyauchi, Saori Hayashi, Takashi Kikukawa, Megumi Kubo, Makoto Demura, Vadivel Ganapathy, Naoki Kamo BIOPHYSICAL JOURNAL 92 (7) 2559 -2569 2007年04月 [査読無し][通常論文]
     
    Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl- pump. To characterize the electrophysiological features of the Cl- pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl- pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl-, Br-, I-, NO3-, and SCN-, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl- current via pHR and the light intensity was linear, demonstrating that transport of Cl- is driven by a single- photon reaction and that the steady- state current is proportional to the excited pHR molecule. The current- voltage relationship for pHR- mediated photoinduced currents was also linear between -150 mV and 150 mV. The slope of the line describing the current- voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl- as the substrate for the anion pump activity of pHR was about -400 mV. The value for VR was independent of light intensity, meaning that the VR re. ects the intrinsic value of the excited pHR molecule. The value of VR changed signi. cantly for the R123K mutant of pHR. We also show that the Cl- pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl- pump. We have also analyzed the kinetics of voltage- dependent Cl- pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl- transport via pHR is presented.
  • FUJITANI Naoki, KOHNO Takahide, NAKAHARA Taku, TAKAYA Kenji, OSAKI Tsukasa, KAWABATA Shun-ichiro, MIZUGUCHI Mineyuki, AIZAWA Tomoyasu, DEMURA Makoto, NISHIMURA Shin-Ichiro, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 239 -239 2007年03月01日
  • KAMIYA Masakatsu, NAKATOGAWA Shin-ichi, ODA Yasunori, KAMIJIMA Tatsuro, AIZAWA Tomoyasu, KUMAKI Yasuhiro, HAYAKAWA Yoichi, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 186 -186 2007年03月01日
  • SHIMAMOTO Satoshi, AIZAWA Tomoyasu, DEMURA Makoto, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 338 -338 2007年03月01日
  • Chisa Hasegawa, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Yuki Sudo, Megumi Kubo, Makoto Demura, Naoki Kamo PHOTOCHEMISTRY AND PHOTOBIOLOGY 83 (2) 293 -302 2007年03月 [査読無し][通常論文]
     
    An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [20061 1274). Previously, we reported that the phR double mutant, P240T/F250Y(phR), can bind with pHtrII. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR -> P-1 -> P2 -> P3 -> P-4 -> phR. The P-3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P-3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation because of the inhibition of formation of the X-intermediate.
  • Chisa Hasegawa, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Yuki Sudo, Megumi Kubo, Makoto Demura, Naoki Kamo PHOTOCHEMISTRY AND PHOTOBIOLOGY 83 (2) 293 -302 2007年03月 [査読無し][通常論文]
     
    An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [20061 1274). Previously, we reported that the phR double mutant, P240T/F250Y(phR), can bind with pHtrII. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR -> P-1 -> P2 -> P3 -> P-4 -> phR. The P-3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P-3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation because of the inhibition of formation of the X-intermediate.
  • Shin Saito, Tomoyasu Aizawa, Kyosuke Kawaguchi, Takeshi Yamaki, Daisuke Matsumoto, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Sigeharu Takiya, Makoto Demura, Keiichi Kawano BIOCHEMISTRY 46 (7) 1703 -1713 2007年02月 [査読無し][通常論文]
     
    Fibroin-modulator-binding protein 1 (FMBP-1) is a factor that binds the transcriptional activation elements of the fibroin gene. It has a novel structure, consisting of four tandem repeats (R1-R4) of 23 amino acids each in the C-terminal half. This region is referred to as the STPR (score and three amino acid peptide repeat) domain and acts as a DNA-binding domain in FMBP-1. Interestingly, the homology among the four repeats is remarkably high. Here, we have determined the three-dimensional structures of the four repeats by NMR. All four repeat units have basically the same structure: a short alpha-helix in the N-terminal half maintained by a salt bridge and an N-capping box. CD studies showed that the full-length STPR domain was 31% helical in solution. This is explained by the connections among the four short helices that were determined separately by NMR. From the thermal-denaturation study, it can be deduced that these four helices in the full-length STPR domain moved flexibly with no interaction among them. However, the specific DNA caused a distinct increase, of up to 76%, in the alpha-helical content of the full-length STPR domain. This finding suggests that the binding of the full-length STPR domain to specific DNA causes an induced-fit conformational change that increases alpha-helicity; the poorly structured regions of the protein may form a regular secondary structure. Furthermore, the mutation analysis showed that the four repeats of the STPR domain raise the possibility of interaction with DNA in different ways.
  • Makoto Takeuchi, Mineyuki Mizuguchi, Takahide Kouno, Yoshinori Shinohara, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Keiichi Kawano PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 66 (3) 716 -725 2007年02月 [査読無し][通常論文]
     
    Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin. Proteins 2007;66:716-725. (c) 2006 Wiley-Liss, Inc.
  • 王 巍, 伊藤 聰一郎, 松田 篤, 相沢 智康, 出村 誠, 四宮 謙一, 田中 順三 末梢神経 = Peripheral nerve 17 (2) 195 -198 2006年12月01日 [査読無し][通常論文]
  • Masanori Yasui, Taku Miyahara, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta PROTEIN JOURNAL 25 (7-8) 475 -482 2006年12月 [査読無し][通常論文]
     
    In order to investigate the thermodynamics of the unfolding of metalloproteins, the thermal denaturation of bovine alpha-lactalbumin (BLA), a typical calcium-binding protein, was investigated under a wide variety of calcium ion activities by means of differential scanning calorimetry. The excess heat capacity obtained as above is composed of those of the following three reactions: (i) the release of a calcium ion from holo-BLA; (ii) the capture of the released calcium ion by the chelating reagent; and (iii) the denaturation of native apo-BLA. The results indicated that the presence of the chelating reagent had a remarkable effect on the apparent enthalpy change for the denaturation of holo-BLA. On the other hand, the influence of the chelator on the heat capacity change was shown to be negligible. Because the denaturation reaction of holo-BLA includes Reactions (i) and (iii), it had to be handled as a three-state reaction. Such an investigation of the unfolding has been scarcely found that the activity of the metal ion is controlled precisely in wide range.
  • Mikihiro Shibata, Yuko Saito, Makoto Demura, Hideki Kandori CHEMICAL PHYSICS LETTERS 432 (4-6) 545 -547 2006年12月 [査読無し][通常論文]
     
    Halorhodopsin (HR) is a light-driven chloride pump in haloarchaea. Our previous low-temperature Fourier-transform infrared (FTIR) study of pharaonis HR reported that the chloride binding site is destabilized in the L-1 intermediate because of the changes in its polar environment. The present FTIR spectroscopy of the wild-type and mutant pharaonis HR revealed that Glu234 becomes deprotonated in the L-2 intermediate. This suggests that appearance of a negative charge at the extracellular surface prevents a chloride ion from translocation toward the extracellular side. (c) 2006 Elsevier B.V. All rights reserved.
  • Joo-Hong Yeo, Kwang-Gill Lee, Hae-Yong Kweon, Soon-Ok Woo, Sang-Mi Han, Sung-Su Kim, Makoto Demura JOURNAL OF APPLIED POLYMER SCIENCE 102 (1) 772 -776 2006年10月 [査読無し][通常論文]
     
    Fractionated components of Bombyx mori silk fibroin, which were hydrolyzed with protease, were prepared with a preparative-recycling high-performance-liquid-chromatography system to evaluate the protective effects of molecular-weight-controlled B. mori silk fibroin components on H2O2-injured neuronal cells. Three major fractions, having molecular weights less than about 1500, were first collected with the recycling techniques. The highest protective effect of the molecular-weight-controlled B. mori silk fibroin components on H2O2-injured neuronal cells was obtained when a fraction with a molecular weight of approximately 1400 was used. This protective effect of the silk fibroin hydrolysate on H2O2-injured neuronal cells was suggested to correlate with the contents of aromatic amino acids such as tyrosine and phenylalanine. (c) 2006 Wiley Periodicals, Inc.
  • Norikazu Muneda, Mikihiro Shibata, Makoto Demura, Hideki Kandori Journal of the American Chemical Society 128 (19) 6294 -6295 2006年05月17日 [査読無し][通常論文]
     
    In the FTIR study of rhodopsins, we have so far found that strongly hydrogen-bonded water molecules (O-D stretch at < 2400 cm-1) are only present in the proteins exhibiting proton-pumping activity. Halorhodopsin (HR) is a light-driven chloride pump in haloarchaea, which does not possess such water molecules. On the other hand, it is known that addition of azide converts HR into a proton pump. Although the mechanism has not been understood, we observed strongly hydrogen-bonded water molecules in the azide-bound HR of Natronobacterium pharaonis (pHR). This finding is consistent with the previous results, implying that the presence of strongly hydrogen-bonded water molecules is requested for the proton-pumping function of rhodopsins. Copyright © 2006 American Chemical Society.
  • Norikazu Muneda, Mikihiro Shibata, Makoto Demura, Hideki Kandori JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 128 (19) 6294 -6295 2006年05月 [査読無し][通常論文]
  • SAITO Shin, MATSUMOTO Daisuke, KAWAGUCHI Kyosuke, YAMAKI Takeshi, AIZAWA Tomoyasu, TAKIYA Shigeharu, KUMAKI Yasuhiro, DEMURA Makoto, NITTA Katsutoshi, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 253 -256 2006年03月01日
  • NMR insight of structural stability and folding of calcium-binding lysozyme
    Modern Magnetic Resonance Part 1: Applications in Chemistry, Biological and Marine Sciences/,493-497 2006年 [査読無し][通常論文]
  • Masahito Tada, Yoshinori Shinohara, Ichiro Kato, Koichi Hiraga, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Mineyuki Mizuguchi, Keiichi Kawano ACTA HISTOCHEMICA ET CYTOCHEMICA 39 (2) 31 -34 2006年 [査読無し][通常論文]
     
    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein ( GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.
  • A Novel N14Y Mutation in Connexin26 in KID Syndrome ? Analyses of Altered Gap Junctional Communication and Molecular Structure of N-Terminus of Mutated Connexin26
    Journal of the American Chemical Society 128 (19) 6294 -6295 2006年 [査読無し][通常論文]
  • Satoshi Watanabe, Masahito Tada, Tomoyasu Aizawa, Masanobu Yoshida, Tadamasa Sugaya, Makoto Taguchi, Takahide Kouno, Takashi Nakamura, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano Protein and peptide letters 13 (8) 815 -822 2006年 [査読無し][通常論文]
     
    GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The H-1 NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core P-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn(2). Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.
  • M Mizuguchi, A Matsuura, Y Nabeshima, K Masaki, M Watanabe, T Aizawa, M Demura, K Nitta, Y Mori, H Shinoda, K Kawano PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 61 (2) 356 -365 2005年11月 [査読無し][通常論文]
     
    M. Mizuguchi, A. Matsuura, Y. Nabeshima, K. Masaki, M. Watanabe, T. Aizawa, M. Demura, K. Nitta, Y. Mori, H. Shinoda and K. Kawano
    Effects of the Stabilization of the Molten Globule State on the Folding Mechanism of alpha-Lactalbumin: A Study of a Chimera of Bovine and Human alpha-Lactalbumin
    Proteins, 61, 356-365 (2005).*
  • 出村 誠 生物物理 45 (1) S7 2005年10月19日 [査読無し][通常論文]
  • 中野 学, 相沢 智康, 三浦 和紀, 星野 宏和, 宮澤 光博, 加藤 祐輔, 熊木 康裕, 出村 誠, 津田 栄, 河野 敬一, 新田 勝利 生物物理 45 (1) S45 2005年10月19日 [査読無し][通常論文]
  • 佐藤 寿哉, 秋元 薫, 神谷 昌克, 相沢 智康, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S45 2005年10月19日 [査読無し][通常論文]
  • 秋元 薫, 佐藤 寿哉, 板橋 実希, 神谷 昌克, 相沢 智康, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S45 2005年10月19日 [査読無し][通常論文]
  • 秋枝 大介, 安井 雅範, 野中 康宏, 島本 怜史, 野積 拓也, 相沢 智康, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S45 2005年10月19日 [査読無し][通常論文]
  • 野中 康宏, 秋枝 大介, 相沢 智康, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S52 2005年10月19日 [査読無し][通常論文]
  • 林 早織, 宮内 正二, 長谷川 千紗, 出村 誠, 加茂 直樹 生物物理 45 (1) S98 2005年10月19日 [査読無し][通常論文]
  • 佐藤 麻希, 菊川 峰志, 加茂 直樹, 相沢 智康, 新田 勝利, 出村 誠, 河野 敬一 生物物理 45 (1) S98 2005年10月19日 [査読無し][通常論文]
  • 久保 恵美, 相沢 智康, 加茂 直樹, 新田 勝利, 河野 敬一, 出村 誠 生物物理 45 (1) S99 2005年10月19日 [査読無し][通常論文]
  • 長谷川 千紗, 須藤 雄気, 下野 和実, 宮内 正二, 出村 誠, 加茂 直樹 生物物理 45 (1) S99 2005年10月19日 [査読無し][通常論文]
  • 斉藤 裕子, 久保 恵美, 佐藤 真希, 菊川 峰志, 相沢 智康, 加茂 直樹, 新田 勝利, 河野 敬一, 出村 誠 生物物理 45 (1) S99 2005年10月19日 [査読無し][通常論文]
  • 齋藤 伸, 八巻 健, 川口 恭輔, 松本 大祐, 山本 宏子, 上島 達郎, 相沢 智康, 滝谷 重治, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S133 2005年10月19日 [査読無し][通常論文]
  • 松本 大祐, 八巻 健, 齋藤 伸, 川口 恭輔, 山本 宏子, 上島 達朗, 相沢 智康, 滝谷 重治, 出村 誠, 新田 勝利, 河野 敬一 生物物理 45 (1) S133 2005年10月19日 [査読無し][通常論文]
  • 仲谷 博安, 中尾 正治, 槙 亙介, 佐伯 喜美子, 相沢 智康, 出村 誠, 河野 敬一, 桑島 邦博 生物物理 45 (1) S136 2005年10月19日 [査読無し][通常論文]
  • 宗田 法和, 柴田 幹大, 佐々木 貴規, 出村 誠, 加茂 直樹, 神取 秀樹 生物物理 45 (1) S188 2005年10月19日 [査読無し][通常論文]
  • 神取 秀樹, 古谷 祐詞, 柴田 幹大, 住井 昌代, 水出 紀子, 宗田 法和, 池田 大亮, 川鍋 陽, 加茂 直樹, 出村 誠, 井原 邦夫, Jung K.-H, Brown L.S 生物物理 45 (1) S189 2005年10月19日 [査読無し][通常論文]
  • 佐々木 貴規, 相沢 智康, 加茂 直樹, 新田 勝利, 河野 敬一, 出村 誠 生物物理 45 (1) S235 2005年10月19日 [査読無し][通常論文]
  • M Kubo, M Sato, T Aizawa, C Kojima, N Kamo, M Mizuguchi, K Kawano, M Demura BIOCHEMISTRY 44 (39) 12923 -12931 2005年10月 [査読無し][通常論文]
     
    Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-D-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-D-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 MM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <-> OG-NpHR (disassembled) <-> intermediate -> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.
  • M Sato, M Kubo, T Aizawa, N Kamo, T Kikukawa, K Nitta, M Demura BIOCHEMISTRY 44 (12) 4775 -4784 2005年03月 [査読無し][通常論文]
     
    Natronomonas (Natronobacterium.) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl- ion pump. For efficient Cl- transport, the existence of Cl-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl- to anion-free NpHR. The binding affinity was affected in Mutants in which certain EC residues had been replaced; this finding revealed the importance of Ar123. On the other hand, Mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC Mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants. L2 -photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl- to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.
  • M. Demura, T. Yoshida, T. Hirokawa, Y. Kumaki, T. Aizawa, K. Nitta, I. Bitter, K. Toth Interaction of dopamine and acetylcholine with an amphiphilic resorcinarene receptor in aqueous micelle system Bioorganic & Medicinal Chemistry Letters, 15, 1367-137・・・
    2005年 [査読無し][通常論文]
     
    M. Demura, T. Yoshida, T. Hirokawa, Y. Kumaki, T. Aizawa, K. Nitta, I. Bitter, K. Toth
    Interaction of dopamine and acetylcholine with an amphiphilic resorcinarene receptor in aqueous micelle system
    Bioorganic & Medicinal Chemistry Letters, 15, 1367-1370 (2005)*
  • T. Sasaki, M. Sonoyama, M. Demura, S. Mitaku Photobleaching of bacteriorhodopsin solubilized with Triton X-100 Photochemistry and Photobiology, 81, 1131-1137 (2005).*
    2005年 [査読無し][通常論文]
  • M. Shibata , N. Muneda , T. Sasaki , K. Shimono, N. Kamo, M. Demura, H. Kandori Hydrogen-Bonding Alterations of the Protonated Schiff Base and Water Molecule in the Chloride Pump of Natronobacterium pharaonis Biochemistry, 44, 12279-12286 (2005).*
    2005年 [査読無し][通常論文]
  • 秋枝 大介, 相沢 智康, 安井 雅範, 野中 康宏, 渡部 正博, 山本 宏子, 出村 誠, 河野 敬一, 新田 勝利 生物物理 44 (1) S30 2004年11月10日 [査読無し][通常論文]
  • 松本 大祐, 八巻 健, 川口 恭輔, 山本 宏子, 熊木 康裕, 相沢 智康, 滝谷 重治, 出村 誠, 新田 勝利 生物物理 44 (1) S30 2004年11月10日 [査読無し][通常論文]
  • 中野 学, 相沢 智康, 三浦 和紀, 星野 宏和, 宮澤 光博, 加藤 祐輔, 熊木 康裕, 出村 誠, 津田 栄, 河野 敬一, 新田 勝利 生物物理 44 (1) S30 2004年11月10日 [査読無し][通常論文]
  • 仲戸川 真一, 織田 康則, 相沢 智康, 出村 誠, 早川 洋一, 新田 勝利 生物物理 44 (1) S44 2004年11月10日 [査読無し][通常論文]
  • 佐々木 貴規, 出村 誠, 相沢 智康, 新田 勝利 生物物理 44 (1) S57 2004年11月10日 [査読無し][通常論文]
  • 佐藤 麻希, 久保 恵美, 相沢 智康, 加茂 直樹, 新田 勝利, 出村 誠 生物物理 44 (1) S58 2004年11月10日 [査読無し][通常論文]
  • 久保 恵美, 佐藤 麻希, 加茂 直樹, 相沢 智康, 出村 誠, 新田 勝利 生物物理 44 (1) S93 2004年11月10日 [査読無し][通常論文]
  • 柴田 幹大, 宗田 法和, 佐々木 貴規, 下野 和実, 出村 誠, 加茂 直樹, 神取 秀樹 生物物理 44 (1) S93 2004年11月10日 [査読無し][通常論文]
  • 宗田 法和, 柴田 幹大, 佐々木 貴規, 下野 和実, 出村 誠, 加茂 直樹, 神取 秀樹 生物物理 44 (1) S93 2004年11月10日 [査読無し][通常論文]
  • 島本 怜史, 相沢 智康, 安井 雅範, 山本 宏子, 出村 誠, 新田 勝利, 河野 敬一 生物物理 44 (1) S196 2004年11月10日 [査読無し][通常論文]
  • 渡部 正博, 相沢 智康, 出村 誠, 新田 勝利 生物物理 44 (1) S196 2004年11月10日 [査読無し][通常論文]
  • 松浦 篤志, 水口 峰之, 鍋島 裕子, 出村 誠, 新田 勝利, 河野 敬一 生物物理 44 (1) S200 2004年11月10日 [査読無し][通常論文]
  • 野中 康宏, 渡部 正博, 相沢 智康, 水口 峰之, 出村 誠, 新田 勝利 生物物理 44 (1) S204 2004年11月10日 [査読無し][通常論文]
  • M Watanabe, T Aizawa, M Demura, K Nitta BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1702 (2) 129 -136 2004年11月 [査読無し][通常論文]
     
    The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degreesC. The DeltaV values at 25 degreesC were obtained for the N-MG (-46 cm(3)/mol) and MG-unfolded-state (U) transition (-40 cm(3)/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm(3)/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state, of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate. (C) 2004 Elsevier B.V. All rights reserved.
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta PROTEIN AND PEPTIDE LETTERS 11 (4) 325 -330 2004年08月 [査読無し][通常論文]
     
    The effect of pressure on the unfolding of the molten globule (MG) state of canine milk lysozyme (CML) was examined Using ultraviolet spectroscopy. The volume changes of the MG-unfolded-state transition were observed at pH 2.0 and around 20 to 60degreesC. but 110 volume change has been found for bovine alpha-lactalbumin, Which is homologous to CML. Our results suggest that the MG state of CML possesses a tightly packed hydrophobic core.
  • M Shibata, N Muneda, K Ihara, T Sasaki, M Demura, H Kandori CHEMICAL PHYSICS LETTERS 392 (4-6) 330 -333 2004年07月 [査読有り][通常論文]
     
    Halorhodopsin and bacteriorhodopsin convert light into energy in archaea through light-driven chloride and proton pumps, respectively. Three water molecules are present in their active centers, which presumably stabilize the quadrupole structures and play crucial roles in pumps. The present low-temperature Fourier-transform infrared (FTIR) study revealed that hydration of the negative charges by the internal water molecules is much weaker in halorhodopsin than in bacteriorhodopsin, suggesting that chloride ion is stabilized by weak hydrogen bonds of waters in halorhodopsin. (C) 2004 Elsevier B.V. All rights reserved.
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta PROTEIN JOURNAL 23 (5) 335 -342 2004年07月 [査読無し][通常論文]
     
    The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.
  • K Arita, M Akiyama, T Aizawa, Segawa, I, D Sawamura, M Demura, K Nitta, H Shimizu JOURNAL OF INVESTIGATIVE DERMATOLOGY 122 (3) A69 -A69 2004年03月 [査読無し][通常論文]
  • M. Yoshida, K. Shitara, K. Matsubara, T. Kouno, T. Aizawa, Y. Hayakawa, Y. Kumaki, M. Mizuguchi, M. Demura, K. Nitta, K. Kawano JOURNAL OF PEPTIDE SCIENCE 10 226 -226 2004年 [査読無し][通常論文]
  • M. Yoshida, T. Aizawa, T. Nakamura, K. Shitara, Y. Hayakawa, K. Matsubara, K. Miura, T. Kouno, K. D. Clark, M. R. Strand, M. Mizuguchi, M. Demura, K. Nitta, K. Kawano, "The Gly-Gly Linker Region of the Insect Cytokine Growth Blocking Peptide (GBP) is E・・・
    2004年 [査読無し][通常論文]
     
    M. Yoshida, T. Aizawa, T. Nakamura, K. Shitara, Y. Hayakawa, K. Matsubara, K. Miura, T. Kouno, K. D. Clark, M. R. Strand, M. Mizuguchi, M. Demura, K. Nitta, K. Kawano, "The Gly-Gly Linker Region of the Insect Cytokine Growth Blocking Peptide (GBP) is Essential for Activity", J. Biol. Chem., 279, 51331-51337 (2004).*
  • M. Shibata, N. Muneda, K. Ihara, T. Sasaki, M. Demura, and H. Kandori, "Internal Water Molecules of Light-Driven Chloride Pump Proteins", Chem. Phys. Lett., 392, 330-333 (2004).*
    2004年 [査読無し][通常論文]
  • 川口 恭輔, 八巻 健, 相沢 智康, 滝谷 重治, 出村 誠, 新田 勝利 生物物理 43 (1) S41 2003年08月25日 [査読無し][通常論文]
  • 吉田 正暢, 設楽 邦夫, 松原 隆英, 河野 隆英, 相沢 智康, 早川 洋一, 熊木 康裕, 水口 峰之, 出村 誠, 新田 勝利, 河野 敬一 生物物理 43 (1) S47 2003年08月25日 [査読無し][通常論文]
  • 安井 雅範, 宮原 卓, 相沢 智康, 出村 誠, 新田 勝利 生物物理 43 (1) S69 2003年08月25日 [査読無し][通常論文]
  • 板橋 実希, 増渕 雄輝, 相沢 智康, 熊木 康裕, 出村 誠, 新田 勝利 生物物理 43 (1) S72 2003年08月25日 [査読無し][通常論文]
  • 出村 誠, 久保 恵美, 佐藤 麻希, 相沢 智康, 加茂 直樹, 新田 勝利 生物物理 43 (1) S183 2003年08月25日 [査読無し][通常論文]
  • 奥田 秀泰, 須藤 雄気, 三島 正規, 佐藤 麻希, 出村 誠, 新田 勝利, 加茂 直樹, 児嶋 長次郎 生物物理 43 (1) S183 2003年08月25日 [査読無し][通常論文]
  • M Sato, T Kikukawa, T Araiso, H Okita, K Shimono, N Kamo, M Demura, K Nitta JOURNAL OF BIOCHEMISTRY 134 (1) 151 -158 2003年07月 [査読無し][通常論文]
     
    Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl-binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl- ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.
  • 奥田秀泰, 須藤雄気, 三島正規, 佐藤麻希, 出村誠, 新田勝利, 加茂直樹, 児嶋長次郎 NMR討論会講演要旨集 42nd 2003年
  • M. Sato, T. Kikukawa, T. Araiso, H. Okita, K. Shimono, N. Kamo, M. Demura, and K. Nitta, "Ser-130 of Natronobacterium pharaonis Halorhodopsin Is Important for the Chloride Binding", Biophys. Chem., 104, 209-216 (2003).*
    2003年 [査読無し][通常論文]
  • N. Fujitani, M. Kanagawa, T. Aizawa, T. Ohkubo, S. Kaya, M. Demura, K. Kawano, K. Taniguchi, K. Nitta, "Structure Determination and Conformational Change Induced by Tyrosine Phosphorylation of the N-terminal Domain of the a-Chain of Pig Gastric H+/K+-A・・・
    2003年 [査読無し][通常論文]
     
    N. Fujitani, M. Kanagawa, T. Aizawa, T. Ohkubo, S. Kaya, M. Demura, K. Kawano, K. Taniguchi, K. Nitta, "Structure Determination and Conformational Change Induced by Tyrosine Phosphorylation of the N-terminal Domain of the a-Chain of Pig Gastric H+/K+-ATPase", Biochem. Biophys. Res. Commun., 300, 223-229 (2003).*
  • M Tada, Y Kobashigawa, M Mizuguchi, K Miura, T Kouno, Y Kumaki, M Demura, K Nitta, K Kawano BIOCHEMISTRY 41 (46) 13807 -13813 2002年11月 [査読無し][通常論文]
     
    Tada, Y. Kobashigawa, M. Mizuguchi, K. Miura, T. Kouno, Y. Kumaki, M. Demura, K. Nitta, K. Kawano, "NMR Stabilization of Protein by Replacement of a Fluctuating Loop: Structural Analysis of a Chimera of Bovine alpha-Lactalbumin and Equine Lysozyme", Biochemistry, 41, 13807-13813 (2002).*
  • 佐藤 麻希, 相沢 智康, 出村 誠, 新田 勝利, 加茂 直樹 生物物理 42 (2) S119 2002年10月10日 [査読無し][通常論文]
  • 武藤 香織, 設楽 邦夫, 吉田 正暢, 飯利 春奈, 松原 公明, 三浦 和紀, 相沢 智康, 水口 峰之, 出村 誠, 早川 洋一, 新田 勝利, 河野 敬一 生物物理 42 (2) S131 2002年10月10日 [査読無し][通常論文]
  • 吉田 正暢, 設楽 邦夫, 松原 公明, 相沢 智康, 早川 洋一, 三浦 和紀, 熊木 康裕, 水口 峰之, 出村 誠, 新田 勝利, 河野 敬一 生物物理 42 (2) S131 2002年10月10日 [査読無し][通常論文]
  • 飯利 春奈, 相沢 智康, 松本 恭子, 早川 洋一, 出村 誠, 新田 勝利, 河野 敬一 生物物理 42 (2) S131 2002年10月10日 [査読無し][通常論文]
  • N Koganesawa, T Aizawa, H Shimojo, K Miura, A Ohnishi, M Demura, Y Hayakawa, K Nitta, K Kawano PROTEIN EXPRESSION AND PURIFICATION 25 (3) 416 -425 2002年08月 [査読無し][通常論文]
     
    N. Koganesawa, T.Aizawa, H.Shimojo, K.Miura, A.Ohnishi, M. Demura, Y.Hayakawa, K.Nitta, K.Kawano, "Expression and purification of small cytokine growth-blocking peptide (GBP) from armyworm Pseudaletia separata by the optimized fermentation method using methylotrophic yeast Pichia pastoris", Protein Expression and Purification, 25, 416-425 (2002).*
  • MORITA Hirokazu, NIIDOME Takuro, MURAKAMI Hiroto, KAWAZOE Masahito, HATAKEYAMA Tomomitu, KOBASIGAWA Yosihiro, MATSUSITA Minako, KUMAKI Yasuhiro, DEMURA Makoto, NITTA Katsutoshi, AOYAGI Haruhiko Peptide science : proceedings of the ... Japanese Peptide Symposium 2001 343 -346 2002年03月01日
  • M Sato, T Kanamori, N Kamo, M Demura, K Nitta BIOCHEMISTRY 41 (7) 2452 -2458 2002年02月 [査読無し][通常論文]
     
    pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N-3(-), which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degreesC was 260s(-1) with in apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (0 --> hR' reaction) reported previously [Varo et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.
  • M. Demura, M. Noda, T. Kajimoto, T. Uchiyama, K. Umemoto, C.-H. Wong, T. Asakura, "Solution Structure of Sialyl Lewis X mimic Studied by Two-dimensional NMR", J.Mol.Struc., 602, 215-222 (2002).*
    2002年 [査読無し][通常論文]
  • M.Mizuguchi, Y. Kobashigawa, Y. Kumaki, M. Demura, K. Kawano and K. Nitta, "Effects of a Helix Stabilization on the Folding Mechanism of alpha-lactalbumin", Proteins, 49, 95-103 (2002).*
    2002年 [査読無し][通常論文]
  • N. Fujitani, S. Kawabata, T. Osaki, Y. Kumaki, M. Demura. K. Nitta and K. Kawano, "Structure of the antimicrobial peptide Tachystatin A", J. Biol. Chem., 277, 23651-7 (2002).*
    2002年 [査読無し][通常論文]
  • T. Miyazaki, T. Kaneko, J.P. Gong, Y. Osada, M. Demura, M. Suzuki, "Water-Induced Crystallization of Hydrogels", Langmuir, 18, 965-967 (2002).*
    2002年 [査読無し][通常論文]
  • K. Sasahara, M. Demura, and K. Nitta, "Equilibrium and Kinetic Folding of Hen Egg-white Lysozyme under Acidic Conditions", Proteins, 49, 472-82 (2002).*
    2002年 [査読無し][通常論文]
  • T.Suetake, T.Aizawa, N.Koganesawa, T.Osaki, Y.kobashigawa, M. Demura, S.Kawabata, K.Kawano, S.Tsuda, and K.Nitta, "Production and Characterization of Recombinant Tachycitin, the Chitin-Binding Protein", Protein Engineering, 15, 763-769 (2002).*
    2002年 [査読無し][通常論文]
  • A. Matsuura, M. Yao, T. Aizawa, N. Koganesawa, K. Masaki, M. Miyazawa, M. Demura, I. Tanaka, K. Kawano, K. Nitta, "Structural Analysis of an Insect Lysozyme Exhibiting Catalytic Efficiency at Low Temperatures", Biochemistry, 41 , 12086 - 12092 (2002).*
    2002年 [査読無し][通常論文]
  • T Koshiba, Y Kobashigawa, M Demura, K Nitta PROTEIN ENGINEERING 14 (12) 967 -974 2001年12月 [査読無し][通常論文]
     
    Thermodynamics of thermal transitions of a calcium-binding lysozyme, canine milk lysozyme (CML), was studied using differential scanning calorimetry and compared with those for homologous proteins, human alpha-lactalbumin (alpha-hLA) and equine milk lysozyme (EML). The results showed that CML and EML exhibit two clear heat absorption peaks in the absence of calcium ions (apo-form), although the cooperative thermal transition of alpha-hLA is apparently absent in this form. The first peak represents the unfolding transition from the native to an unfolding intermediate state (N-I transition) and the second peak represents that from the intermediate to the thermally unfolded state (I-U transition). We interpret that the cooperative thermal transition, which is observed between the intermediate and the thermally unfolded states of CML and EML, comes from the native-like packing interaction in their intermediate states. Furthermore, to examine the role of the stabilization mechanism of CML intermediate, we constructed four variant CMLs (H21G, I56L, A93S and V109K), in which the residues of CML are substituted for those of EML, and also investigated their thermal stability. Especially the His21 and Val109 of CML play a role in stabilization of the intermediate state and their contributions to the unfolding free energy are estimated to be 2.0 and 1.8 kJ/mol, respectively. From the results of the mutational analysis, a few differences in the local helical interactions within the alpha-domain are found to be predominant in stabilizing the intermediate state.
  • 相沢 智康, 吉田 正暢, 設楽 邦夫, 松原 公明, 早川 洋一, 三浦 和紀, 小金澤 望, 藤谷 直樹, 熊木 康裕, 水口 峰之, 出村 誠, 新田 勝利, 河野 敬一 生物物理 41 (1) S102 2001年09月10日 [査読無し][通常論文]
  • 渡部 正博, 小橋川 敬博, 山本 宏子, 小柴 琢己, 出村 誠, 新田 勝利 生物物理 41 (1) S164 2001年09月10日 [査読無し][通常論文]
  • Y Kurokawa, N Koganesawa, Y Kobashigawa, T Koshiba, M Demura, K Nitta JOURNAL OF PROTEIN CHEMISTRY 20 (4) 293 -303 2001年05月 [査読無し][通常論文]
     
    Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) approximate to SS3(Cys65-Cys81) > SS4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D-helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.
  • 藤谷直樹, 川畑俊一郎, 尾崎司, 熊木康裕, 岩永貞昭, 出村誠, 新田勝利, 河野敬一 日本薬学会年会要旨集 121st (4) 115 2001年03月05日 [査読無し][通常論文]
  • AIZAWA Tomoyasu, HOSHINO Hirokazu, FUJITANI Naoki, KOGANESAWA Nozomi, MATSUURA Atsushi, MIYAZAWA Mitsuhiro, KATO Yusuke, KUMAKI Yasuhiro, DEMURA Makoto, NITTA Katsutoshi, KAWANO Keiichi Peptide science : proceedings of the ... Japanese Peptide Symposium 2000 269 -272 2001年03月01日
  • 出村 誠 生物物理 41 (1) 20 -23 2001年01月25日 [査読無し][通常論文]
  • Binding Feature of Mastoparan Inducing Phase Change of Lipid Bilayers
    生物物理 41 20 -23 2001年 [査読無し][通常論文]
  • Y. Kobashigawa, K. Miura, M. Demura, N. Nemoto, T. Koshiba, K. Nitta, and S. Tsuda: "Assignment of 1H, 13C, and 15N Resonances of Canine Milk Lysozyme", Journal of Biomolecular NMR, 19: 387-388 (2001).*
    2001年 [査読無し][通常論文]
  • T. Aizawa, Y. Hayakawa, A. Ohnishi, N. Fujitani, K. D. Clark, K. Miura, N. Koganesawa, Y. Kumaki, M. Demura, M. R. Strand, K. Nitta and K. Kawano: "Minimized structure of small insect cytokine, growth-blocking peptide: essential region for mitogenic an・・・
    2001年 [査読無し][通常論文]
     
    T. Aizawa, Y. Hayakawa, A. Ohnishi, N. Fujitani, K. D. Clark, K. Miura, N. Koganesawa, Y. Kumaki, M. Demura, M. R. Strand, K. Nitta and K. Kawano: "Minimized structure of small insect cytokine, growth-blocking peptide: essential region for mitogenic and hemocyte stimulating activities are different", J. Biol. Chem., 276: 31813-31818 (2001).*
  • Y. Hori, M. Demura, M. Iwadate, A. S. Ulrich, T. Niidome, H. Aoyagi, and T. Asakura: "Interaction of Mastoparan with Membranes Studied by 1H-NMR in Detergent Micelles and by Solid State 2H-NMR and 15N-NMR in Oriented Lipid Bilayers", Eur. J. Biochem., ・・・
    2001年 [査読無し][通常論文]
     
    Y. Hori, M. Demura, M. Iwadate, A. S. Ulrich, T. Niidome, H. Aoyagi, and T. Asakura: "Interaction of Mastoparan with Membranes Studied by 1H-NMR in Detergent Micelles and by Solid State 2H-NMR and 15N-NMR in Oriented Lipid Bilayers", Eur. J. Biochem., 267: 302-309 (2001).*
  • T. Niidome, H. Murakami, M. Kawazoe, T. Hatakeyama, Y. Kobashigawa, M. Matsushita, Y. Kumaki, M. Demura, K. Nitta, and H. Aoyagi: "Carbohydrate Recognition of Gramicidin S Analogs in Aqueous Medium", Bioorganic Medicinal Chemistry Letters, 11: 1893-18・・・
    2001年 [査読無し][通常論文]
     
    T. Niidome, H. Murakami, M. Kawazoe, T. Hatakeyama, Y. Kobashigawa, M. Matsushita, Y. Kumaki, M. Demura, K. Nitta, and H. Aoyagi: "Carbohydrate Recognition of Gramicidin S Analogs in Aqueous Medium", Bioorganic Medicinal Chemistry Letters, 11: 1893-1896 (2001).*
  • Y Kobashigawa, M Demura, T Koshiba, Y Kumaki, K Kuwajima, K Nitta PROTEINS-STRUCTURE FUNCTION AND GENETICS 40 (4) 579 -589 2000年09月 [査読無し][通常論文]
     
    The native state H-1, N-15 resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states, On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state. (C) 2000 Wiley-Liss, Inc.
  • 黒川 洋輔, 小橋川 敬博, 小柴 琢己, 出村 誠, 新田 勝利 生物物理 40 (1) S5 2000年08月05日 [査読無し][通常論文]
  • 相沢 智康, 藤谷 直樹, 三浦 和紀, 小金澤 望, 早川 洋一, 大西 敦, 熊木 康裕, 出村 誠, 新田 勝利, 河野 敬一 生物物理 40 (1) S31 2000年08月05日 [査読無し][通常論文]
  • 小橋川 敬博, 水口 峰之, 出村 誠, 小柴 琢己, 津田 栄, 新田 勝利 生物物理 40 (1) S160 2000年08月05日 [査読無し][通常論文]
  • K Sasahara, M Demura, K Nitta BIOCHEMISTRY 39 (21) 6475 -6482 2000年05月 [査読無し][通常論文]
     
    Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.
  • M Osanai, M Okudaira, J Naito, M Demura, T Asakura INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 30 (3) 225 -232 2000年03月 [査読無し][通常論文]
     
    The derivation of alanine in fibroin was investigated using NMR and selective isotopic labelling. (H2O)-H-2 infused orally into 5th instar larvae was incorporated into the proton of the methyl group of alanine in fibroin. Proton exchange among alanine, glycine and serine was also found. Incorporation of C-13 from [2-C-13]acetate into alanine C2 and C3 and glycine C2 in fibroin, and also C4 of free glutamine plus glutamate was observed in vivo. Hemolymph contained a peak for C4 of glutamate plus glutamine, and an alanine C3 peak appeared transiently. Thus, it is suggested that the C-skeleton of alanine formed was derived from L-malate via the TCA-cycle, and that this alanine is utilized in part for fibroin synthesis. Spectra of the hemolymph extract of larvae infused orally with [N-15(2)]urea showed no 1(5N)-compounds, whereas those of larvae injected subcutaneously showed only one peak of urea, whose intensity decreased with time, as shown in the in vivo spectra of a living larva infused with [N-15(2)]urea. The solution NMR spectrum of fibroin showed no N-15-labelled compounds. Temporal changes in the peak intensities of six compounds in the spectra of a Living larva infused with [N-15]ammonium demonstrated a process in which N-15 was incorporated into fibroin containing N-15-alanine through the amide group of glutamine and the amino group of glutamate. Thus, alanine biosynthesis from the. TCA-cycle originates mainly from water, L-malate and ammonium. The fact that no N-15-urea was detected in the hemolymph extract of larvae infused with [N-15]ammonium suggests that N-15-urea found in the above in vivo spectra may be that accumulated in the hindgut. Thus, excess ammonium in the body causes the production of urea by the urea-cycle. In Samia larvae, urea was not reutilized but excreted. The metabolic relationships between the assimilation of ammonium and the function of the urea-cycle are discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.
  • T Ito, Y Maruhashi, M Demura, T Asakura POLYMER 41 (3) 859 -866 2000年02月 [査読無し][通常論文]
     
    The structure of uniaxially oriented poly(L-lactic acid) (PLLA) with different drawn ratios was studied in the solid state using C-13 NMR spectroscopy. The spectra of uniaxially oriented PLLA films were observed by changing the angle between the draw direction of the samples and the magnetic field. The observed spectra of the samples with draw ratios of 3 and 4 were well reproduced by the simulated spectra, indicating the presence of two kinds of oriented components and one unoriented component in these samples. The angles, theta(C=O), between the C=O bond direction of PLLA and the oriented axis of the chain were calculated to be 132 and 128 degrees for two oriented components, respectively, by assuming that the chemical shift tensor direction relative to the molecular symmetry axis of the carbonyl carbon nucleus of PLLA was the same as that for the carbonyl carbon of poly(ethylene terephthalate) reported previously. The angles, 132 and 128 degrees, are in agreement with the reported angles for PLLA 3(l) helix model in the beta form and PLLA 10(3) helix model in the alpha form, respectively. The relatively small value of p (characterizing the distribution of the fiber axis) obtained in the simulation indicates the high orientation in the oriented domain in the PLLA film samples. The fraction of each component in the samples was also determined. (C) 1999 Elsevier Science Ltd. All rights reserved.
  • M. Mizuguchi, K. Masaki, M. Demura and K. Nitta: "Local and Long-range Interactions in the Molten Globule State: a study of chimeric proteins of bovine and human alpha-lactalbumin", J. Mol. Biol., 298, 985-995 (2000)*
    2000年 [査読無し][通常論文]
  • J.D.van Beek, L. Beaulieu, H. Schafer, M. Demura, T. Asakura and B.H. Meier: "Solid-state NMR determination of the secondary structure of Samia cynthia ricini silk", Nature, 405, 1077-1079 (2000)*
    2000年 [査読無し][通常論文]
  • T. Koshiba, M. Yao, Y. Kobashigawa, M. Demura, A. Nakagawa, I. Tanaka, K. Kuwajima and K. Nitta: "Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme", Biochemistry, 39, 3248-3257 (2000)*
    2000年 [査読無し][通常論文]
  • 小橋川敬博, 小柴琢己, 出村誠, 藤谷直樹, 熊木康裕, 引地邦男, 河野敬一, 桑島邦博, 新田勝利 NMR討論会講演要旨集 38th 107 -108 1999年09月30日 [査読無し][通常論文]
  • 出村 誠, 増渕 雄輝, 長岡 悟志, 堀 由美子, 朝倉 哲郎, 新田 勝利 生物物理 39 (1) S87 1999年09月02日 [査読無し][通常論文]
  • 相沢 智康, 小金沢 望, 松浦 篤志, 鈴木 政人, 星野 宏和, 田谷内 政人, 藤谷 直樹, 宮澤 光博, 加藤 祐輔, 熊木 康裕, 出村 誠, 河野 敬一, 引地 邦男, 新田 勝利 生物物理 39 (1) S103 1999年09月02日 [査読無し][通常論文]
  • 水口 峰之, 正木 和夫, 出村 誠, 新田 勝利 生物物理 39 (1) S163 1999年09月02日 [査読無し][通常論文]
  • Higuchi, A., Uchiyama, S., Demura, M.: Asakura, T., Cho, C.-S., Akaike, T., Takarada, T. and Hara, M., "Enhanced CEA production associated with aspirin in a culture of CW-2 cells on some polymeric films", Cytotechnology, 31: 233-242 (1999)*
    1999年 [査読無し][通常論文]
  • Hori, Y., Demura, M., Niidome, T., Aoyagi, H., Asakura, T.: " Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR", FEBS Lett., 455: 228-232 (1999)*
    1999年 [査読無し][通常論文]
  • Asakura, T. and Demura, M.: "Structure and Dynamics of Silk Fibroin Studied with 13C, 15N and 2H Solid State NMR", Macromol., Macromolecular Symposia, 143: 1-10 (1999)*
    1999年 [査読無し][通常論文]
  • Asakura, T., Iwadate, M., Demura, M. and Williamson, M.P.: " Structural analysis of silk with 13C NMR chemical shift contour plots", International Journal of Biological Macromolecules, 24: 167-171 (1999)*
    1999年 [査読無し][通常論文]
  • T Ohno, M Hara, M Demura, T Asakura, A Higuchi ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 216 U291 -U291 1998年08月 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠 高分子 47 (6) 390 -393 1998年06月01日 [査読無し][通常論文]
     
    シルクは,繊維の女王として数千年にわたる衣料への利用の歴史があるが,優れた繊維性能はもとより,多岐にわたる優れた特性をもっており,それを生かした応用研究もまた進みつつある.本稿では,シルク利用の基礎となるその構造について得られつつある最新の知見と未解決な点,さらに非繊維分野へのシルク利用の展開を中心に概説する.
  • T Asakura, Y Yamazaki, KW Seng, M Demura JOURNAL OF MOLECULAR STRUCTURE 446 (3) 179 -190 1998年05月 [査読無し][通常論文]
     
    Asakura,T., Yamazaki,Y., Koo, W.-S. and Demura,M.: "Determination of the Mutual Orientation of the 15N and 13C NMR Chemical Shift Tensors of 13C-15N Double Labeled Model Peptides for Silk Fibroin from the Dipolar-Coupled Powder Patterns", J.Molecular Structure, 446:179-190(1998)
  • 出村 誠, 朝倉 哲朗 高分子 47 (6) 402b -402b 1998年 [査読無し][通常論文]
  • Demura,M., Yamazaki,Y., Asakura, T. and Ogawa,K.: "Structure of Uniaxially Aligned 13C Labeled Silk Fibroin Fibers with Solid State 13C-NMR", J.Molecular Structure, 441:155-163(1998)*
    1998年 [査読無し][通常論文]
  • Asakura, T. and Demura,M.: "Oriented Polymers" in Solid State NMR of Polymers (I. Ando and T. Asakura Eds.) Elsevier, 307-326 (1998)
    1998年 [査読無し][通常論文]
  • Asakura,T., Demura,M., Yoshimizu, H. and Nishikawa,N.: "Proteins" in Solid State NMR of Polymers (I. Ando and T. Asakura Eds.) Elsevier, 853-890(1998)
    1998年 [査読無し][通常論文]
  • 出村 誠, 斉田 理, 笹平 理朗, 石坂 弘子, 朝倉 哲郎, Meier B.H 生物物理 37 (2) S14 1997年09月05日 [査読無し][通常論文]
  • T Asakura, M Minami, R Shimada, M Demura, M Osanai, T Fujito, M Imanari, AS Ulrich MACROMOLECULES 30 (8) 2429 -2435 1997年04月 [査読無し][通常論文]
     
    Three kinds of H-2-labeled Bombyx mori silk fibroin samples (with [2,2-H-2(2)]Gly, [3,3,3-H-2(3)]Ala, or [2,3,5,6-H-2(4)]Tyr) were obtained by oral administration of either the labeled amino acid or (H2O)-H-2 to 5th instar larvae. The administration of (H2O)-H-2 alone yielded a high degree of selective deuteration at the alanine methyl group, since the incorporation of (H2O)-H-2 occurs between fumarate and malate in the tricarboxylic acid (TCA) cycle of the silk fibroin synthetic pathway. Uniaxially oriented silk fibers were prepared as samples for H-2-NMR spectroscopy. An analysis of the quadrupole echo line shape was carried out in order to determine the angle of the deuterium-labeled group relative to the fiber axis, i.e., of the C alpha-H-2 bond vectors in glycine and of C alpha-C beta(2)H(3) in alanine. With the fiber axis aligned parallel to the magnetic field, quadrupole splittings were obtained as 117.8 and 39.8 kHz for [2,2-H-2(2)]Gly- and [3,3,3-H-2(3)]Ala-labeled silk, respectively. These values are identical with those obtained from the 2H-NMR powder patterns of the unaligned samples, within experimental error. From the angular dependence of the quadrupole splittings, it was thus calculated that the C alpha-H-2 bonds of glycine as well as the C alpha-C beta(2)H(3) bond of alanine make an angle of approximately 90 degrees relative to the fiber axis. These steric constraints were then used to evaluate the torsion angles, phi and psi, for the glycine and alanine residues within the protein backbone. These data, determined independently by solid-state H-2 NMR, thus verified and narrowed down the allowed region in the Ramachandran (phi, psi) map obtained from previous solid-state C-13- and N-15-NMR studies.
  • ASAKURA T. Biopolymers 41 (2) 193 -203 1997年 [査読無し][通常論文]
  • T. Asakura, M. Demura, N. Naoki Annual Reports on NMR Spectroscopy) (Ed. G.A. Webb) (ACADEMIC PRESS, London) 34 301 -346 1997年 [査読無し][通常論文]
  • Refinement of the Solution Structure of Neurokinin A (4-10) on the Basis of 1H NMR Chemical Shift Calculations
    T. Asakura, M. Iwadate, T. Date, M. Demura, K. Nokihara Peptide Chemistry 1996 345 -348 1997年 [査読無し][通常論文]
  • Side Chain Dynamics of [3,3-2H2]Serine Labeled Silk Fibroin using Solid State 2H NMR
    Makoto Demura, Yohei Ohkawa, Tetsuo Asakura Rep. Prog. Polym. Phys. Jpn. 40 613 -616 1997年 [査読無し][通常論文]
  • Determination of Molecular Orientation in Peptide-Phospholipid Bilayer System using Angular Dependent Solid State 31P, 2H and 15N NMR
    Yumiko Hori, Makoto Demura, Tetsuo Asakura, Takuro Niidome, Haruhiko Aoyagi Rep. Prog. Polym. Phys. Jpn. 40 609 -612 1997年 [査読無し][通常論文]
  • T Asakura, T Konakazawa, M Demura, T Ito, Y Maruhashi POLYMER 37 (10) 1965 -1973 1996年05月 [査読無し][通常論文]
     
    The structure of uniaxially oriented poly(ethylene terephthalate) (PET) with different draw ratios was studied in the solid state using C-13 nuclear magnetic resonance (n.m.r.) spectroscopy. All of the carbonyl carbons of the samples were isotopically labelled with C-13, and therefore a detailed C-13 n.m.r. analysis, including the determination of the chemical-shift tensor for the carbonyl carbons, was possible. The chemical-shift tensor direction relative to the molecular symmetry axis was determined with the aid of semiempirical quantum-chemical calculations by using the FPT-INDO (finite perturbation theory/intermediate neglect of differential overlap) method. The spectra of uniaxially oriented PET films observed by changing the angle between the draw direction and the magnetic field were analysed. Two components, one oriented and the other unoriented, were detected in the spectra of samples with draw ratios of 3 and 4. A highly oriented component was observed in addition to these two for samples having draw ratios of 5 (before and after heat treatment) and 5.66. The fraction of each component was determined by computer simulation of spectra that had been obtained as a function of sample orientation angle in the magnetic field. The angle theta between the phenylene para C-C axis and the chain axis was 16 degrees +/- 10 degrees and 29 degrees +/- 5 degrees for the oriented components, which is in agreement (within experimental errors) with the values reported previously by others. The angle-dependent spectra simulated with small p (characterizing the distribution of the fibre axis) confirm the presence of a highly oriented structure in the samples. Copyright (C) 1996 Elsevier Science Ltd.
  • Dynamics and Conformation of Uniaxially Oriented Silk Fibroin Fiber Studied with Solid State 2H NMR
    M. Demura, M. Minami, T. Asakura, A. Ulrich Rep. Prog. Polym. Phys. Jpn. 39 613 1996年 [査読無し][通常論文]
  • Determination of the Structure of Sialyl Lewis X Mimetics by 2D-NMR
    T. Asakura, M. Noda, O. Saita, M. Demura, K. Umemoto, T. Kajimoto, C.-H. Wong Rep. Prog. Polym. Phys. Jpn. 39 609 1996年 [査読無し][通常論文]
  • 31P and 15N Solid State NMR Spectra of A Peptide with β-Sheet Structure in Lipid Bilayer Oriented with Glass Plates
    M. Demura, Y. Mizoguchi, T. Asakura Rep. Prog. Polym. Phys. Jpn. 39 617 1996年 [査読無し][通常論文]
  • T. Asakura, A. Aoki, T. Date, M. Demura Polymer J. 28 (1) 24 -29 1996年 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠, Cross T.A 日産科学振興財団研究報告書 (19) 35 -38 1996年 [査読無し][通常論文]
  • 岩舘満雄, 菊地淳, 出村誠, 朝倉哲郎, 藤田憲一 高分子学会予稿集 44 (11) 2913 -2914 1995年09月 [査読無し][通常論文]
  • 岩舘満雄, 菊地淳, 出村誠, 朝倉哲郎 繊維学会シンポジウム予稿集 1995 S.64 1995年06月 [査読無し][通常論文]
  • 朝倉哲郎, 菊地淳, 富谷俊樹, 野田政稔, 出村誠, 大和久光治, 蓮田勝美 高分子学会予稿集 44 (4) 655 1995年05月 [査読無し][通常論文]
  • 新繊維科学ーニューフロンティアへの挑戦
    朝倉哲郎, 出村誠 通商産業調査 475 -479 1995年 [査読無し][通常論文]
  • 2H Labeling of Silk and 2H NMR Analysis of Oriented Silk Fiber
    T. Asakura, M. Minami, R. Shimada, M. Demura Rep. Prog. Polym. Phys. Jpn. 38 605 -608 1995年 [査読無し][通常論文]
  • The Relationship between Amide Proton Chemical Shifts and Secondary Structure in Proteins
    Tetsuo Asakura, Kazuhiro Taoka, Makoto Demura, Michael P. Williamson J. Biomol. NMR 6 227 -236 1995年 [査読無し][通常論文]
  • 朝倉哲郎, 菊地淳, 出村誠, WILLIAMSON M NMR討論会講演要旨集 33rd 361 -364 1994年11月 [査読無し][通常論文]
  • TA CROSS, KC LEE, W HU, RR KETCHEM, S HUO, LK NICHOLSON, JH YEO, M DEMURA, T FUJITO, M IMANARI, T ASAKURA ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 208 109 -PMSE 1994年08月 [査読無し][通常論文]
  • 菊地淳, 萩原健一, 朝倉哲郎, 出村誠, 大和久光治, 伊藤隆男, 蓮田勝美, 藤田憲一 繊維学会シンポジウム予稿集 1994 S.56 1994年06月 [査読無し][通常論文]
  • 菊地淳, 萩原健一, 出村誠, 朝倉哲郎, 大和久光治, 伊藤隆男, 蓮田勝美, 藤田憲一 高分子学会予稿集 43 (4) 1468 1994年05月 [査読無し][通常論文]
  • Tetsuo Asakura, Makoto Demura, Tetsuo Hayashi Annual Reports on NMR Spectroscopy 29 (C) 325 -404 1994年01月01日 [査読無し][通常論文]
     
    This chapter discusses the 13C NMR assignments of polyolefines and olefin copolymers in the solution based on 13C NMR chemical shift calculations and 2D incredible natural abundance double quantum transfer experimen methods (INADEQUATE). Emphasis is placed on revealing the primary structures, such as stereochemical configuration, branching, regioregularity, and comonomer sequence. Two approaches for the assignment of the 13C NMR spectra of polymers are discussed in this chapter. One is 13C NMR chemical shift calculations on the basis of the γ-effect on the 13C chemical shift, using the RIS (rotational isomeric state) model and the other is the 2D INADEQUATE method. The calculation of 13C chemical shifts on the basis of the γ-effects and the RIS model have also been successfully applied to the 12C NMR tacticity assignments of other polymers, such as poly(viny1 chloride) and polystyrene. These calculations clarify the origin of the peak splitting due to the tacticity of the polymer in solution and provide methods to examine the tacticity dependent- and time-averaged local conformations calculated with the RIS model. The solid-state 13C NMR spectra of polyolefines are also well interpreted in terms of the γ-effect. On the other hand, another method, 2D INADEQUATE is especially useful for the sequence analysis of polyolefines and, hereafter, this will be used for the tacticity assignments in addition to the chemical shift calculations. © 1994 Academic Press Limited
  • 生体高分子「高分子材料の機器分析事例集」
    出村誠, 朝倉哲郎 技術情報協会 229 -265 1994年 [査読無し][通常論文]
  • The Origin of The Amide Proton Chemical Shifts of Proteins
    Tetsuo Asakura, Kazuhiro Taoka, Makoto Demura, Michael P.Williamson Reports on Progress in Polymer Physics in Japan 37 713 -714 1994年 [査読無し][通常論文]
  • T ASAKURA, A AOKI, M DEMURA, JM JOERS, RC ROSANSKE, T GULLION POLYMER JOURNAL 26 (12) 1405 -1408 1994年 [査読無し][通常論文]
  • Structural Characterization of Samia cynthis ricini Silk Fibroin as Biomaterial
    Makoto Demura, Tetsuo Asakura Int. J. Wild Silkmoth & Silk 1 (2) 139 -141 1994年 [査読無し][通常論文]
  • 13C NMR Assignments of Polyolefins and Olefine Copolymers Based on the 13C NMR Chemical Shift Calculation and 2D INADEQUATE NMR
    Tetsuo Asakura, Makoto Demura, Tetsuo Hayashi Annual Reports on NMR Spectroscopy 29 325 -404 1994年 [査読無し][通常論文]
  • Joo-Hong Yeo, Makoto Demura, Tetsuo Asakura, Teruaki Fujito, Mamoru Imanari, Linda K. Nicholson, Timothy A. Cross Solid State Nuclear Magnetic Resonance 3 (4) 209 -218 1994年 [査読無し][通常論文]
  • Atsusi Ono, Seizi Miyauchi, Makoto Demura, Tetsuo Asakura, Naoki Kamo Biochemistry 33 (14) 4312 -4318 1994年 [査読無し][通常論文]
  • NMR Characterization of Silk Proteins, ACS Symposium Series; Silks; Biology, Structure, Properties, Gentics
    Tetsuo Askaura, Makoto Demura, Atuso Uyama, Katsuaki Ogawa, Keiichi Ogawa, Linda K. Nicholson, Timothy A. Cross Gentics, American Chemical Society Books Department 544 (13) 148 -154 1994年 [査読無し][通常論文]
  • ASAKURA Tetsuo, DEMURA Makoto, HIRAISHI Yoshiaki, OGAWA Katsuaki, UYAMA Atsuo Chemistry letters 1994 (12) 2249 -2252 1994年 [査読無し][通常論文]
  • Minami Masashi, Takatsu Rika, Demura Makoto, Asakura Tetsuo 繊維学会誌 50 (11) 498 -504 1994年 [査読無し][通常論文]
     
    We have recorded several kinds of 13C NMR to characterize the structure of the noncrystalline domain in Bombyx mori silk fibroin and the influence of hydration on the structure of silk fibroin. First, the structural analysis of the soluble fraction (noncrystalline domain) after chymotrypsin hydrolysis of the silk fibroin has been performed by 13C solution NMR spectroscopy. In the noncrystalline fraction. Cs fraction, the C-terminal amino acid residues are identified as---Gly-Tyr from the pH titration of the spectra. Similarly, the N-terminal residues are identified as Gly-Ser---. Second, the 13C CP/MAS NMR spectra for the precipitated fraction (Cp fraction; crystalline domain) and Cs fraction have been observed as a function of moisture regain in order to know what parts, residue or groups, interact with the adsorbed water. In the Cp fraction, the significant spectral changes were not observed with increasing water content. However, in the Cs fraction, the significant changes were observed at the carbonyl carbon peak of the N-terminal Gly residue and the aromatic Cγ carbon peak of the C-terminal Tyr residue by hydration. Third, the 1H NMR spin-lattice relaxation times, T1 of water in Cp and Cs fractions were observed. The T1 values were analyzed as two components for the Cp fraction and three components for the Cs fraction. The water molecules with the restricted motion increase considerably in the Cs fraction and such a restricted motion is roughly divided into two cases. This seems to be corresponding to the strongly hydration at the terminal groups observed in the 13C CP/MAS NMR spectrum of the Cs fraction.
  • The Contour Plots of the Conformation-Dependent 13C NMR Chemical Shifts of Proteins
    Tetsuo Asakura, Toshiki Tomiya, Takeshi Date, Makoto Demura, Michael P.Williamson Reports on Progress in Polymer Physics in Japan 37 707 -708 1994年 [査読無し][通常論文]
  • 呂 奏洪, 出村 誠, 小中沢 岳仁, 朝倉 哲郎, 伊藤 卓郎, 藤戸 輝昭, 今成 司 高分子論文集 51 (1) 47 -51 1994年 [査読無し][通常論文]
     
    三種のポリアミド系繊維, 絹フィブロイン, poly (p-phenylene-terephthalamide) (PPTA), poly (mphenylene-isophthalamide) (PMIA) の配向ブロック試料を作製し, 磁場に平行と垂直な<SUP>15</SUP>N CP NMRスペクトルを測定した. 前報で誘導したポリアミド系繊維構造解析のための理論式を用いて, 構造パラメーターを決定した. 特に, 非晶部の定量的な構造解析が本手法を用いて可能であった.
  • 北村 愛夫, 陳 開利, 柴本 秋男, 出村 誠 日本蚕糸学雑誌 63 (5) 415 -419 1994年 [査読無し][通常論文]
     
    有機溶媒処理による絹の凝集構造変化について密度, 内部表面積および非結晶領域の分子間水素結合エネルギーオーダー分布 (いわゆるL. O. 分布) の解析から追究した。さらにこの処理絹の構造の安定性について, 水浸処理を追加しての凝集構造からも検討した。この結果有機溶媒処理により, 非結晶領域に構造弛緩がおこり, 微小な空隙が発生していることが分かり, 内部表面積の増大さらにL. O. 分布において, 中, 高相対湿度領域で大きくなり, この領域で破断される水素結合の多いことが明らかとなった。
  • T ASAKURA, JH YEO, M DEMURA, T ITOH, T FUJITO, M IMANARI, LK NICHOLSON, TA CROSS MACROMOLECULES 26 (24) 6660 -6663 1993年11月 [査読無し][通常論文]
  • LK NICHOLSON, T ASAKURA, M DEMURA, TA CROSS BIOPOLYMERS 33 (5) 847 -861 1993年05月 [査読無し][通常論文]
     
    Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state N-15-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from N-15 chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The N-15-H-1 dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies.
  • Synthesis of 13C-15N Double Labeled Sequence Model Compounds of Bombyx mori Silk Fibroin and the 15N and 13C solid State NMR
    Tetsuo Asakura, Yasunobu Yamazaki, Koo Wey Seng, Makoto Demura, Isao Ando Reports on Progress in Polymer Physics in Japan 36 633 -636 1993年 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠 高分子加工 41 (9) p442 -446 1992年09月 [査読無し][通常論文]
  • 出村誠, 平川叙夫, 朝倉哲郎, 伊藤卓郎, 谷川征男, 飯塚英策 日本膜学会年会講演要旨集 14th 1992年
  • L NICHOLSON, T CROSS, M DEMURA, T ASAKURA FASEB JOURNAL 6 (1) A341 -A341 1992年01月 [査読無し][通常論文]
  • Improved Parameters for 1H Chemical Sift Calculation of Proteins
    Tetsuo Asakura, Yosimi Niizawa, Makoto Demura Rep.Prog.Polm. Phys. Jpn. xxxv 655 -658 1992年 [査読無し][通常論文]
  • Tetsuo Asakura, Nobuhiko Nakayama, Makoto Demura, Atsushi Asano Macromolecules 25 (19) 4876 -4881 1992年 [査読無し][通常論文]
  • 出村誠, 平川叙夫, 朝倉哲郎, 谷川征男, 伊藤卓郎, 藤戸輝昭, 飯塚英策 高分子学会予稿集 XXXV (3) 551 -552 1992年 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠, 中村 英二, 安藤 勲 高分子論文集 49 (4) 281 -287 1992年 [査読無し][通常論文]
     
    Basic Pancreatic Trypsin Inhibitor (BPTI) のα-CHとNHプロトンの<SUP>1</SUP>H NMR化学シフトをX線回折より報告されている原子座標に基づいて, 理論的に計算した. α-CHプロトン化学シフトは, 環電流効果とカルボニル基の磁気異方性効果の和として, また, NHプロトン化学シフトは, これらの二つの効果に, さらに反磁性項を加えて評価した. BPTIのα-CH, ならびにNHプロトン化学シフトの計算値は, いずれも実測値をおおむね再現することができた. さらに, 化学シフト計算に基づいて, 溶液中では, Lys46のNHプロトンの関与した水素結合が存在することを指摘した. X線回折の結果では, この水素結合の存在は, 報告されていないが, 溶液のNMR研究では, 対照的にその存在が報告されてきた. このように, 化学シフト計算によってBPTI溶液と固体間の局所的構造の違いを明らかにすることができた.
  • 北村 愛夫, 陳 開利, 柴本 秋男, 出村 誠 日本蚕糸学雑誌 61 (3) 215 -220 1992年 [査読無し][通常論文]
     
    絹の有機溶媒中での伸長・収縮挙動は合成繊維に見られる収縮のみを示すような単純な挙動でなく, その温度依存性から, 伸長のみを示すタイプ (メタノル, ブタノール), 低温においてわずかに伸長挙動を示すが, 高温になると収縮を示し, 概して収縮が支配的なタイプ (ベンゼン, プロパノール, ジオキサン, 四塩化炭素, トリクロロエチレン) および収縮から伸長に転ずるタイプ (エタノール, DMF, DMSO, DMAc) の3のタイプに分けらることを明らかにした。またこの伸長・収縮挙動は測定温度と測定時間に関連する溶媒の侵入速度と分子間の緩和程度に大きく依存していることがわかった。
  • 出村 誠, 竹之下 仁子, 朝倉 哲郎, 酒井 治利, 栗岡 聡, 小松 計一, 金子 正夫 日本蚕糸学雑誌 61 (1) 66 -72 1992年 [査読無し][通常論文]
     
    絹セシリン単独あるいは絹セシリンと絹フィブロインのブレンド水溶液を用いて, 絹フィブロイン不織布にグルコースオキシンダーゼ (GOD) をコーティングし, 固定化されたGODの活性を調べた。絹セシリンのみを用いてGODを不織布にコーティングしたところ, 0.5%のグルタルアルデヒドで処理したにもかかわらず, 絹セシリンのコーティング層からの溶出は, 完全に抑えられなかった。しかしながら, 従来の絹フィブロインでGODをコーティングした場合と同様に, 高活性を示すことがわかった。一方, 絹セシリンを絹フィブロインとともにコーティングした場合, 絹セシリンの溶出はほとんど見られなかった。さらに, 絹セシリンの重量分率が0.14のとき, 絹フィブロインのみの場合より活性が約1.2倍向上した。この現象は, 絹セシリンの重量分率の増加に伴うGODの有量ならびに酸素透過性の増加, ならびに絹フィブロインの分子運動の変化によるためと結論した。
  • 西川 昭文, 嶋崎 暢子, 久保田 静男, 朝倉 哲郎, 出村 誠 日本シルク学会誌 1 (0) S51 -S52 1992年 [査読無し][通常論文]
  • T ASAKURA, E NAKAMURA, H ASAKAWA, M DEMURA JOURNAL OF MAGNETIC RESONANCE 93 (2) 355 -360 1991年06月 [査読無し][通常論文]
  • T ASAKURA, M DEMURA, Y NISHIYAMA MACROMOLECULES 24 (9) 2334 -2340 1991年04月 [査読無し][通常論文]
     
    Assignments of the C-13 NMR spectra of five polyolefins, poly(1-pentene), poly(1-hexene), poly(1-heptene), poly(1-octene), and poly(1-nonene), to tacticity were performed from the C-13 NMR chemical shift calculation on the basis of the C-13 NMR gamma effect and application of the rotational isomeric state model. The origin of the complex spectral behavior of the methylene backbone carbon, C1, among a series of poly-olefins including polypropylene and poly(1-butene) was also successfully interpreted through the chemical shift calculation. The mechanisms of olefin polymerization were analyzed from the values of pentad tacticities obtained from the C3 carbon peak of the side chain on the basis of the bicatalytic sites model. The relative area of hexad peaks calculated with the three parameters optimized in the bicatalytic sites model was also used in the assignment of the C1 carbon peak.
  • T ASAKURA, K HIRANO, M DEMURA, K KATO MAKROMOLEKULARE CHEMIE-RAPID COMMUNICATIONS 12 (4) 215 -220 1991年04月 [査読無し][通常論文]
  • Tetsuo Asakura, Makoto Demura, Mariko Nagashima, Ryuji Sakaguchi, Minoru Osanai, Katsuaki Ogawa Insect Biochem. 21 (7) 743 -748 1991年 [査読無し][通常論文]
  • DEMURA M. J. Membr. Sci. 59 (1) 39 -52 1991年 [査読無し][通常論文]
  • Membrane Potential of Bombyx mori Silk Fibroin Membrane Induced by an Immobilized Enzyme Reaction,
    Makoto Demura, Takashi Komura, Tetsuo Asakura Bioelectrochem. Bioenerg. 26 167 -175 1991年 [査読無し][通常論文]
  • 朝倉 哲郎, 北口 源啓, 出村 誠, 酒井 治利, 金子 正夫, 栗岡 聡, 小松 計一 日本蚕糸学雑誌 60 (6) 466 -474 1991年 [査読無し][通常論文]
     
    種々の不織布にコーティングされた家蚕絹フィブロインおよびグルコースオキシダーゼ (GOD) の分子運動, およびGOD固定化量の決定をESR法で行なうために, スピンラベル家蚕絹フィブロインおよびスピンラベルGODを作製した。絹フィブロイン, ビスコースレーヨン, 6-ナイロン, ポリエチレンテレフタレート, ポリプロピレンの各不織布にコーティングされたスピンラベル絹フィブロインゲルのESRスペクトルパターンは, ほとんど同じであったが, 絹フィブロインで不織布にコーティングしたスピンラベルGODのスペクトルパターンは, 繊維素材に依存した。さらに, 絹フィブロインの2w/v%のスピンラベルGODを使ってコーティングした場合, この酵素固定化率は, 不織布繊維素材によって9から85%まで変化したが。固定化に使用した絹フィブロインゲルの付着量の変化とは対応しなかった。ESRの結果にもとづいて, GODは, 絹フィブロインゲル内で不均一に分布していると結論した。
  • 北村 愛夫, 柴本 秋男, 出村 誠 日本蚕糸学雑誌 59 (1) p43 -48 1990年02月 [査読無し][通常論文]
  • グルコースセンサーへの不織布の応用、7, , 1990.
    朝倉哲郎, 出村誠 バイオインダストリ 7 676 -682 1990年 [査読無し][通常論文]
  • Tetsuo Asakura, Makoto Demura, Hidejiro Ogawa, Kazuhiro Matsushita, Mamoru Imanari Macromolecules 24 (2) 620 -622 1990年 [査読無し][通常論文]
  • 北村 愛夫, 柴本 秋男, 出村 誠 日本蚕糸学雑誌 59 (1) 43 -48 1990年 [査読無し][通常論文]
     
    絹と有機溶媒との相互作用の本質が絹～有機溶媒間の極性, 親和力あるいは親和力に関係する溶解度パラメーター (S. P.) に関連するものであるとの考え方より, 膨潤現象および拡散現象を中心として分子間凝集力緩和の機構を解明しようとした。その結果, 希釈熱 (κ1) は体積膨潤度 (Qv), 拡散定数の対数と密接な関係をもち, κ1の小さい溶媒ほどQvが増加し, 絹に近い S. P. 値をもつ溶媒ほど膨潤力が大きく, κ1の減少にともなって拡散定数の増加することがわかった。また3次元溶解度パラメーターの概念を適用し, 絹の有機溶媒による膨潤に極性力が支配的であることがわかった。
  • 出村 誠, 香村 恭史, 平出 啓, 朝倉 哲郎 繊維学会誌 46 (9) 391 -396 1990年 [査読無し][通常論文]
     
    Lipase derived from Rhizopus delemar was entrapped in the silk fibroin membrane with the methanol-immersion treatment for 10 min. No leakage of the immobilized enzyme was detected by the method using the enzymatic assay. Thermal effect on the activity of immobilized lipase was studied in detail. The optimum temperature for immobilized enzyme (50°C) was higher than that for the free one (20°C). The lipase was much stabilized by the immobilization with silk fibroin since the inactivation constant, which was determined from the measurement of inactivation process at 50°C, was less than 1/40 of that of the free enzyme. The optimum pH shifted to alkaline side as compared to that of the free one. On the basis of several properties concerning the immobilized lipase and dissolubilization state of the substrate, tributyrin, observed by 13C NMR measurement, it was concluded that origin of the stabilization of lipase with silk fibroin was mainly the formation of the unique structure such as Silk II type crystalline in the silk membrane. The lipase-immobilized silk fibroin membrane generated membrane potential with the immobilized-enzyme reaction in the membrane.
  • 朝倉 哲郎, 出村 誠 繊維学会誌 45 (6) P252 -P257 1989年 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠 繊維学会誌 45 (6) Pp252 -257 1989年 [査読無し][通常論文]
  • T ASAKURA, M DEMURA, M TSUTSUMI MAKROMOLEKULARE CHEMIE-RAPID COMMUNICATIONS 9 (12) 835 -839 1988年12月 [査読無し][通常論文]
  • 朝倉 哲郎, 出村 誠 繊維学会誌 44 (11) p535 -540 1988年11月 [査読無し][通常論文]
  • T ASAKURA, Y KAWAGUCHI, M DEMURA, M OSANAI INSECT BIOCHEMISTRY 18 (6) 531 -538 1988年 [査読無し][通常論文]
  • Makoto Demura, Aio Kitamura, Akio Shibamoto, Tetsuo Asakura J.Appl.Polym.Sci. 36 (3) 535 -543 1988年 [査読無し][通常論文]
  • Aio Kitamura, Akio Shibamoto, Makoto Demura Sen'i Gakkaishi 44 (4) 193 -198 1988年 [査読無し][通常論文]
     
    The relationship between the preparation conditions and the function of silk fibroin membrane was investigated in terms of water-sorption, membrane potential, ion permeability, X-ray diffraction and birefringence. We clarified the influences of preparation conditions of the silk fibroin membrane such as the concentration of fibroin solution (dilute and concentrated), substrate (acrylic resin and mercury) and drying rate (slow and rapid), on membrane potential and ion permeability of the fibroin membrane. The orientation function of the amorphous region is remarkably dependent on the preparation conditions, whereas that of crystal region is almost independent. When the mercury was used as a substrate and the drying rate is slow, the aggregation state of the fibroin membrane estimated from the iso-electric point, IEP was the highest. The membrane prepared from a concentrated solution on mercury at the slow-drying rate has the smallest average pore radius [formula omitted]. On the other hand, those for the membranes prepared from a dilute solution on an acrylic sheet are generally larger. It was clarified that the change of ion permeability of the membrane generally agreed to the order of IEP and [formula omitted] of the membrane. © 1988, The Society of Fiber Science and Technology, Japan. All rights reserved.
  • Tetsuo Asakura, Makoto Demura Sen'i Gakkaishi 44 (11) 535 -540 1988年 [査読無し][通常論文]
     
    The physical state and orientation of the water trapped in porous Bombyx mori silk fibroin membranes were examined in the absence and presence of alkali metal salts, NaCl, KCl and LiCI by means of NMR spectroscopy. In spite of the high content of water (93%) in the membrane, doublet 1H NMR peaks of the water molecules were observed and its separation depended strongly on the angle, θ, between the membrane surface and the magnetic field. This trend was independent of the presence of alkali metal ions in the water. From the relationship between the separation of the doublet and the angle, θ, the orientation of the proton-proton axis of the water molecule was found, on the average, parallel to the surface of the membrane. 1H NMR peaks of the water and also, 23Na NMR peaks of the NaCl aqueous solution trapped in the membrane shifted to lower field with increasing angle, θ, indicating the anisotropy of the silk fibroin membrane itself. The diamagnetic susceptibility of the membrane was calculated to be -0.64ppm. © 1988, The Society of Fiber Science and Technology, Japan. All rights reserved.
  • 北村 愛夫, 柴本 秋男, 出村 誠 繊維学会誌 44 (4) 193 -198 1988年 [査読無し][通常論文]
     
    The relationship between the preparation conditions and the function of silk fibroin membrane was investigated in terms of water-sorption, membrane potential, ion permeability, X-ray diffraction and birefringence. We clarified the influences of preparation conditions of the silk fibroin membrane such as the concentration of fibroin solution (dilute and concentrated), substrate (acrylic resin and mercury) and drying rate (slow and rapid), on membrane potential and ion permeability of the fibroin membrane. The orientation function of the amorphous region is remarkably dependent on the preparation conditions, whereas that of crystal region is almost independent. When the mercury was used as a substrate and the drying rate is slow, the aggregation state of the fibroin membrane estimated from the iso-electric point, IEP was the highest. The membrane prepared from a concentrated solution on mercury at the slow-drying rate has the smallest average pore radius [formula omitted]. On the other hand, those for the membranes prepared from a dilute solution on an acrylic sheet are generally larger. It was clarified that the change of ion permeability of the membrane generally agreed to the order of IEP and [formula omitted] of the membrane. © 1988, The Society of Fiber Science and Technology, Japan. All rights reserved.
  • T ASAKURA, M DEMURA, K YAMAMOTO, R CHUJO POLYMER 28 (6) 1037 -1040 1987年05月 [査読無し][通常論文]

特許

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  • 神経再生チューブ及びその製造方法
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  • Method for Producing Denatured Substance of Lactalbumin
    20090192296
  • Method for Producing Denatured Substance of Lactalbumin
    PCT Patent 2007/088869
  • ＮＭＲシグナルの帰属方法
    2007-255910
  • 安定同位体元素を有するポリペプチド混合物の合成方法
    2007-254295
  • Biocatalyst entrapped in a silk fibroin membrane
    United States Patent 4,999,295
  • 生体基質濃度測定装置
    特開平02-262046
  • 生体触媒固定化膜，その製造方法及び該生体触媒固定化膜を用いた生体触媒センサ
    特開平01-231889
  • Biocatalyst entrapped in a silk fibroin membrane
    United States Patent 4,999,295

受賞

  • 2023年02月 北海道大学 教育研究総長表彰
     
    受賞者: 出村 誠
  • 2016年02月 北海道大学 教育総長賞（優秀賞）
     
    受賞者: 出村 誠
  • 1992年 繊維学会 論文賞
     
    受賞者: 出村 誠
  • 1990年 高分子学会 研究奨励賞
     
    受賞者: 出村 誠

共同研究・競争的資金等の研究課題

  • NMR共用プラットフォーム（代表：理研）実施機関：北海道大学（先端NMR ファシリティ NMR 装置）
    文部科学省：先端研究基盤共用促進事業（共用プラットフォーム形成支援プログラム）
    研究期間 : 2016年 -2020年 
    代表者 : 出村 誠
  • 学会等助成(第57回NMR討論会)
    伊藤医薬学術交流財団：
    研究期間 : 2018年09月 -2018年09月 
    代表者 : 出村 誠
  • ソフトマターグローバルステーション・次世代物質生命科学研究センター機器共用ユニッ ト(仮称)
    文部科学省：先端研究基盤共用促進事業（新たな共用システム導入支援プログラム）
    研究期間 : 2016年 -2018年 
    代表者 : 出村 誠, 龔 剣萍
     
    https://www.jst.go.jp/shincho/program/sinkyoyo.html 先端研究基盤共用促進事業（新たな共用システム導入支援プログラム）平成28年度選定機関 北海道大学・ソフトマター機器共用ユニットの運営責任者
  • 光エネルギーによる環境浄化微生物の設計
    文部科学省：科学研究費補助金 挑戦的萌芽研究
    研究期間 : 2016年 -2018年 
    代表者 : 出村 誠
  • 高精度タンパク質固体NMRの圧縮センシング測定法開発
    文部科学省：科学研究費補助金（新学術領域研究(研究領域提案型)）
    研究期間 : 2016年 -2017年 
    代表者 : 出村 誠
  • 文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))
    研究期間 : 2014年 -2015年 
    代表者 : 出村 誠
  • 先端NMRファシリティの共用促進プログラム
    文部科学省：先端研究基盤共用・プラットフォーム形成事業【取組1】先端研究基盤の共用促進
    研究期間 : 2013年 -2015年 
    代表者 : 出村 誠
  • 文部科学省：科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2011年 -2012年 
    代表者 : 出村 誠
     
    光誘起膜電位発生膜タンパク質ハロロドプシン(HR)が神経細胞ネットワークの革新的光制御技術として期待されているが、光刺激効率の向上、ハロロドプシン３量体安定化と機能変調を解明することが重要であると予測された。本研究では、こららの課題を解決することを目的とした。初年度は、系統的な変異体作製とその分光学的特性の分子レベルの解析を計画し、以下の成果が得られた。（１）ハロロドプシンNpHR変異体の極大吸収波長(λmax)の系統的解析 2種類のハロロドプシンNpHR, HsHRのアラインメント相同性比較およびNpHR, HsHR結晶構造座標データから、HR機能に重要な残基を推定した。この構造科学的予測からNpHR変異体（25アミノ酸残基点変異, 2ループ領域の部分削除体）を大腸菌発現系により作製した。機能型タンパク質として得られる試料について、可視光波長領域の吸収スペクトル観測を行い、吸収極大波長λmaxの変化を解析することができた。特にループ領域の変異体試料データの系統的解析によって、λmaxシフトが反転する異常シフトを発見した。（２）HR変異体の静的構造安定性評価 実験（１）で得られた試料からλmaxシフトを示す候補についてCl解離定数の決定、可視CD測定による３量体形成能の測定、退色速度から構造安定性実験を実施した。特にループ領域の系統的部分削除体のλmaxシフトが反転する異常シフトは、ループ長の構造安定性とも強い相関があることが解明された。次年度はさらに本研究で作製された試料についてフラッシュフォトリシスによる可視光レーザーパルスによる過渡的吸収変化のフォトアクティビティ解明し、静的構造安定性との関係解明が課題として絞り込まれた。
  • 文部科学省：科学研究費補助金(特定領域研究)
    研究期間 : 2006年 -2007年 
    代表者 : 出村 誠, 相沢 智康, 河野 敬一, 神谷 昌克
     
    リゾチーム・αラクトアルブミンのフォールディングに関する基礎データを活かし、水溶性タンパク質のフォールディング挙動と、その構造中間体が示す細胞膜結合機能との関連を分子レベルで解明することを目的としている。昨年度までに、新規複合体調製法の開発、酸処理法で得られた脂肪酸結合型αラクトアルブミンのアポトーシス誘導活性等に関する成果が得られ、今年度新たに以下の成果が得られた。(1)酸処理法と熱処理法で得られた脂肪酸結合型ウシαラクトアルブミンの生理的条件下での近紫外CDスペクトルは、オレイン酸濃度に依存して三次構造崩壊を示唆する楕円率低下が起こる。NMRでも脂肪酸結合型ウシαラクトアルブミンは非天然構造型スペクトルパターンを示した。脂肪酸結合型ウシαラクトアルブミンの1H NMRスペクトルおよび15N HSQCスペクトルでは、脂肪酸結合による化学シフト変化はαラクトアルブミンのαドメインで大きく、しかもβドメイン側に面するアミノ酸残基であることがわかった。(2)αβαの3つのドメインからなるヒトとウシ由来のαラクトアルブミン(HHHおよびBBB)のドメインを入れかえたキメラ体(HBB,BHB,BBH)との比較から、アポトーシス誘導に関与しているドメインについて考察した。熱処理法で得られたサンプルではMG状態での安定性とアポトーシス活性の強い相関があった。(3)αラクトアルブミンの天然構造の示す水溶性と基質選択性補因子機能は、Molten Globule様構造を酸性処理という簡便な方法で脂肪酸トラップすることができ、細胞死誘導活性という新規の生物機能に変換することができた。今後、αラクトアルブミンと脂肪酸による複合体のNMRキャラクタリゼーションをさらに詳細に検討することで、構造中間体のトラップとそのダイナミクス情報が得られると期待できる。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2004年 -2006年 
    代表者 : 出村 誠, 新田 勝利, 相沢 智康, 河野 敬一, 新田 勝利
     
    α-lactalbumin(α-LA)はミルクに含まれる123残基からなるカルシウム結合蛋白質で、β-1,4-ガラクトース転移酵素の基質選択性の補因子機能をもつ。α-LAの構造は天然状態において、N末端側1-34残基とC末端側86-123残基にα-helical構造を形成し、中間の35-85残基はβ-sheet構造を持つ。低pHやカルシウムフリーの条件下では折りたたみ中間体(MG状態)を形成する。一方、ヒトミルクから単離されたオレイン酸-α-LA複合体が癌細胞死を誘導する新たな機能が報告された。しかし、生物種ごとのα-LAのMG状態形成との関係や、α-LAの活性化部位、オレイン酸結合部位などの詳細は不明である。本研究ではαラクトアルブミンとオレイン酸の混合溶液に熱処理を施すことで複合体が得られること、また酸性Molten Globule状態のαラクトアルブミンとオレイン酸の混合溶液から複合体が得られることを発見し、複合体形成工程がより簡便になった。これらの手法を用いて調製した複合体について腫瘍細胞を用いたアポトーシスの活性評価を行った。トリパンブルー及びMTS cell proliferation assayの結果、アポトーシス誘導後6時間以内にL1210細胞の約90%が死滅することが明らかになった。本研究をとおして、αラクトアルブミンが既知のβ-1,4-ガラクトース転移酵素の基質選択性の補因子機能をもつこと以外に、αラクトアルブミンとオレイン酸の特殊な複合体(変性体)が生物学的に重要なアポトーシス誘導型αラクトアルブミンの創出できる成果が得られた。
  • 文部科学省：科学研究費補助金(特定領域研究)
    研究期間 : 2004年 -2005年 
    代表者 : 新田 勝利, 出村 誠, 出村 誠, 相沢 智康, 新田 勝利, 河野 敬一
     
    α-ラクトアルブミン(α-LA)はミルクに含まれる123残基からなるカルシウム結合蛋白質で、β-1,4-ガラクトース転移酵素の基質選択性の補因子機能をもつ。α-LAの構造は天然状態において、N末端側1-34残基とC末端側86-123残基にα-helical構造を形成し、中間の35-85残基はβ-sheet構造を持つ。低pHやカルシウムフリーの条件下では折りたたみ中間体(MG状態)を形成する。一方、ヒトミルクから単離されたオレイン酸-α-LA複合体が癌細胞死を誘導する新たな機能が報告された。しかし、生物種ごとのα-LAのMG状態形成との関係や、α-LAの活性化部位、オレイン酸結合部位などの詳細は不明である。本年度は昨年度に引き続き、本研究ではホモロジーの高いヒト型α-LA(HLA)、ウシ型α-LA(BLA)およびこれらの遺伝子組み換え体を用い、各種脂肪酸-α-LA複合体の調整方法の考案と細胞死誘導活性を評価した。調整方法として従来よりもより簡便な熱処理法を考案した。この方法は、従来法より多くの利点をもつので、特許出願した。この方法により、牛乳からアポトーシス誘導型調整試料を作製できた。
  • 生体高分子のナノ構造体形成と応用
    研究期間 : 2003年
  • 絹遺伝子の転写制御関連タンパク質
    研究期間 : 2001年
  • ミルクαラクトアルブミンのアポトーシス誘導型構造特性
    研究期間 : 1999年
  • ハロロドプシンのオリゴマー形成
    研究期間 : 1999年
  • タンパク質のNMR化学シフト
    研究期間 : 1997年
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 1995年 -1995年 
    代表者 : 出村 誠
     
    同位体ラベル法と固体NMR法を用い、その利点を生かして高活性を有する状態でのメリチンやグラミシジンAのような膜ペプチドの原子レベルでの立体構造解析をするためには、配向した脂質二重層中に膜ペプチドを埋め込み配向を制御する必要がある。本研究では脂質二重層の配向系を作製し、化学シフトが線形を支配するためにリン脂質の状態に線形が極めて敏感である31Pを測定核として、固体31PNMRによりそれをモニターした。ジミリストイルホスファチジルコリン(DMPC)を有機溶媒に溶解、それをガラスプレート上に均一に広げ、サンプルホルダー中に積み重ねた。約60%に水和し、両端をシリコン栓とエポキシ系の接着剤を用いて密封し、45°Cに保ったインキュベータ-中に放置し、配向試料とした。配向試料についてガラス面(脂質のアルキル分子はガラス面に垂直)と磁場方向が垂直もしくは平行となるように設定して固体31PNMR(JEOL GX400 NMR測定モードGated high power decoupling)を測定した。その結果、約60%に水和したDMPCの無配向パウダーパターン(isoは等方ピーク)、約一週間、45°Cに保った配向試料をガラス面が磁場BOに垂直になるように設定し測定、およびガラス面が磁場BOに平行になるように設定し測定した各固体31PNMRスペクトルのピークは、乾燥粉末(運動なし)のパウダーパターンと大きく異なり、アルキル分子軸を中心として回転する軸対象運動を行っていることをを示していること、また、主ピークの位置は著しく異なり、それぞれは(1)におけるσ||σ⊥の値に対応した。このことより本実験条件にて作成された配向試料は、おおむねDMPCのアルキル鎖がガラス面に垂直に配向しており、軸対称運動を行っていることがわかった。さらに、この配向脂質膜に、モデルペプチドを導入することによって、脂質中のペプチド配向を直接観測することに成功した。
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 1994年 -1994年 
    代表者 : 出村 誠
     
    膜電位プローブの分配される環境である生体膜のモデルとして、ジパルミトイルフォスファチジルコリン(DMPC)、ジパルミトイルフォスファチジルコリン(DPPC)、および卵黄レシチンを使い、グラスプレート間に挿入されたこれらの脂質の配向状態を調べた。特に、固体NMR特有の化学シフトテンソルによる構造情報から定量的に脂質分子の配向角度を決定できることが明かとなった。すなわち、脂質極性部にあるP-31原子の化学シフトテンソルを測定することにより、脂質分子の取りうる様々な相構造変化をモニターできるとともに、その配向挙動の確立分布を計算できることも分かってきた。このような解析方法が確立されてきたので、膜電位プローブの配向挙動を解析する最善の実験手法は、そのプローブに存在するP-31原子の化学シフトテンソルの解析である。また、生体膜の膜電位発生機構のなかで、膜電位発生膜タンパク質が注目されており、今後、脂質膜-膜電位プローブ-膜タンパク質の3成分系に関する固体NMR研究が益々重要となってくると考えられる。
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 1993年 -1994年 
    代表者 : 出村 誠
     
    研究代表者は、これまでに、タンパク質の二次構造形成能に着目し、このタンパク質膜中に外来の酵素を導入することによって、酵素固定化材料の開発を行ってきた。この方法の特徴は、担体となるタンパク質の自己組織的な水素結合網(二次元結晶)形成能を利用することで、酵素等を従来の方法より安定に包括できることである。これは、タンパク質膜に分子認識能を付与する機能化の一貫であったが、その研究過程で、特殊な酵素反応(刺激)による物質移動によって、そのタンパク質膜が電気化学的な応答(膜電位)を示すことを新たに見いだした。本研究では、上記のような反応と拡散の共役した膜系おける膜電位発生機構を明らかにすることを目的とし、特に、タンパク質の配向構造のキャラクタリゼーションを試みた。酵素固定化機能のある絹フィブロインタンパク質の一軸配向試料を作製し、その^<15>Nおよび^<13>固体NMR測定をした。ここで用いた解析手法は、ペプチド平面のNHまたはNC'結合の配向軸に対する角度とその配向分布が決定できる方法を応用した。その結果、絹フィブロインの結晶性領域と非晶性領域フラクションの決定、各々の領域に含まれるGly,Ala,Ser,Tyr,Valの配向角度、核残基の主鎖内部二面体角(phi、psi)、水分吸着によるペプチド結合の動的な構造変化などを定量的に決定することができた。これらの原子レベルの結晶構造に関する知見は、さらに、電気化学的な特性と相関づけることによって、膜電位発生機構解明へと発展させることが可能と考えられる。
  • NMRによる膜タンパク質・GPCRの構造と機能の研究、プロテイン・インフォマティクス
  • NMR of membrane protein and GPCR, Protein informatics

教育活動情報

主要な担当授業

  • ソフトマター解析学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 分光法、NMR、細胞膜、タンパク質科学、バイオマテリアル、機能性ポリマー、プレゼンテーション
  • 生命情報分子科学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 分光法、NMR、細胞膜、タンパク質科学、バイオマテリアル、機能性ポリマー、プレゼンテーション
  • ソフトマター設計学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : インフォマティクス, プログラミング, 確率統計, 機械学習, バイオインフォマティクス, ゲノムデータ
  • 生命情報分子科学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : インフォマティクス, プログラミング, 確率統計, 機械学習, バイオインフォマティクス, ゲノムデータ
  • ソフトマター科学概論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • 生命融合科学概論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • 大学院共通授業科目（一般科目）：複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : インターンシップ、キャリア形成、ビジネススキル、人脈形成、すべての仕事に必要な基本スキル、企業における研究開発、リーダーシップ、グローバル人材
  • 大学院共通授業科目（一般科目）：複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 企業　研究開発　博士　就職　PD　DC キャリアパス多様化　マッチング
  • 大学院共通授業科目（一般科目）：複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 企業　研究開発　実際の業務　キャリアパス　多様性
  • 大学院共通授業科目（一般科目）：複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : キャリアマネジメント（Career Management）、　研究マネジメント（Research Management）、すべての仕事に必要な基本スキル（Transferable Skills）、イノベーション　（Innovation）、マーケティング（Marketing）、知的財産（Intellectual Property）、プレゼンテーション（Presentation）、アントレプレナーシップ（Entrepreneurship）、発想法（Method of thinking）、企業における研究開発（Industrial R&D）、リーダーシップ（Leadership）、グローバル人材（Global Human Resource）
  • 生物系の分光学
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 光と分子、分光法、スペクトル、分子構造、可視紫外吸収スペクトル、赤外線吸収スペクトル、ラマンスペクトル、蛍光スペクトル
  • 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ)
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 持続可能な開発目標(SDGs)、生命科学、科学技術イノベーション
  • 細胞構造科学Ⅰ
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 細部膜、脂質、膜タンパク質、エネルギー変換、物質輸送、生物機能の応用、アクティブラーニング、SDGs

大学運営

学内役職歴

  • 2013年4月1日 - 2015年3月31日 教育研究評議会評議員
  • 2015年4月1日 - 2017年3月31日 教育研究評議会評議員
  • 2017年4月1日 - 2019年3月31日 教育研究評議会評議員
  • 2022年4月1日 - 2024年3月31日 研究戦略室室員
  • 2020年4月1日 - 2022年3月31日 人材育成本部副本部長
  • 2021年4月1日 - 2022年3月31日 総長補佐
  • 2022年4月1日 - 2024年3月31日 総長補佐
  • 2013年4月1日 - 2015年3月31日 大学院先端生命科学研究院長
  • 2015年4月1日 - 2017年3月31日 大学院先端生命科学研究院長
  • 2017年4月1日 - 2019年3月31日 大学院先端生命科学研究院長
  • 2019年4月1日 - 2021年3月31日 大学院先端生命科学研究院副研究院長
  • 2021年4月1日 - 2023年3月31日 大学院先端生命科学研究院副研究院長

委員歴

  • 2023年 - 現在   SDGs QUEST みらい甲子園 北海道大会実行委員会   実行委員長
  • 2021年07月 - 現在   北海道   北海道SDGs連携会議委員
  • 2020年 - 2022年   日本核磁気共鳴学会   理事
  • 2016年 - 2019年   日本核磁気共鳴学会   評議員
  • 2016年01月 - 2017年03月   大学評価・学位授与機構   国立大学教育研究評価委員会専門委員
  • 2012年 - 2014年   日本キチン・キトサン学会   理事
  • 2012年 - 2013年   日本生物物理学会   運営委員
  • 2009年 - 2010年   日本生物物理学会北海道支部   支部長
  • 2008年 - 2009年   日本生物物理学会   編集副委員長
  • 2008年 - 2009年   日本核磁気共鳴学会   理事
  • 2005年 - 2007年   日本生物物理学会   運営委員   日本生物物理学会
  • 2002年 - 2003年   日本化学会北海道支部   幹事
  • 2002年 - 2003年   日本生物物理学会   地区編集委員   日本生物物理学会

社会貢献活動

  • SDGs QUEST みらい甲子園 北海道大会実行委員会・実行委員長
    期間 : 2023年 - 現在
    役割 : 運営参加・支援
  • 北海道大学 x SDGs
    期間 : 2022年04月21日
    役割 : 講師
    主催者・発行元 : 大和証券株式会社
    イベント・番組・新聞雑誌名 : 北海道の未来を拓くSDGs～企業経営と次世代の教育～
  • 先端科学移動大学（市民講座）
    期間 : 2020年11月14日 - 2020年11月14日
    役割 : 講師
    主催者・発行元 : 一般財団法人 北海道青少年科学文化財団
    イベント・番組・新聞雑誌名 : 第28回先端科学移動大学
  • 先端科学移動大学（高等学校訪問授業）
    期間 : 2020年11月13日
    役割 : 講師
    主催者・発行元 : 一般財団法人 北海道青少年科学文化財団
    イベント・番組・新聞雑誌名 : 第28回先端科学移動大学
  • 期間 : 2019年11月16日
    役割 : 講師
    主催者・発行元 : 一般財団法人 北海道青少年科学文化財団
    イベント・番組・新聞雑誌名 : 第28回先端科学移動大学
    社会人・一般
  • 先端科学移動大学（高等学校訪問授業）
    期間 : 2019年11月15日
    役割 : 講師
    主催者・発行元 : 一般財団法人 北海道青少年科学文化財団
    イベント・番組・新聞雑誌名 : 第28回先端科学移動大学
  • 学際研究交流会&SDGs勉強会 in 北大 vol.3
    期間 : 2019年04月17日
    役割 : 講師
    主催者・発行元 : 北大人材育成本部
  • 期間 : 2019年03月18日
    役割 : パネリスト
    主催者・発行元 : 北大サステイナブルキャンパスマネジメント本部
  • 期間 : 2018年12月06日
    役割 : 講師
    主催者・発行元 : 北大先端生命科学研究院・理学研究院

メディア報道

  • SDGs×北海道農業〜Sustainable Hokkaido
    報道 : 2022年06月
    発行元・放送局 : FM NorthWave
    番組・新聞雑誌 : L2nd
    合同企画「Sustainable Hokkaido」(FM NorthWave, AIR-G’)　テレビ・ラジオ番組
  • 北海道農業とSDGs
    報道 : 2022年02月
    発行元・放送局 : HBC
    番組・新聞雑誌 : あぐり王国北海道NEXT
    　テレビ・ラジオ番組

その他

  • 研究論文の研究分野とSDGsとの相関分析 
    [SDGs 3 : 4 : 7 = 60 : 20: 20]（database: 調整中）

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