基本情報

所属

• 先端生命科学研究院

職名

• 准教授

学位

• 博士（工学）（北海道大学）

ホームページURL

https://life.sci.hokudai.ac.jp/fa/staff/kikukawa-takashi

科研費研究者番号

• 20281842

ORCID ID

•  https://orcid.org/0000-0002-6185-7281

J-Global ID

• 200901098397303517

研究キーワード

• 多剤排出蛋白質   過渡吸収分光   膜輸送   膜蛋白質   レチナール蛋白質   Bio-Physical Chemistry   Photo-Biology   

研究分野

• ナノテク・材料 / 生物分子化学
• ライフサイエンス / 薬系分析、物理化学
• ライフサイエンス / 生物物理学

担当教育組織

•  学士課程　理学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2021年06月 - 現在 北海道大学 先端生命科学研究院 准教授
• 2013年04月 - 2021年05月 北海道大学 先端生命科学研究院 講師
• 2011年 北海道大学 先端生命科学研究院 助教

学歴

• 1993年04月 - 1996年03月   北海道大学   大学院工学研究科   生体工学専攻博士後期課程
• 1991年04月 - 1993年03月   北海道大学   大学院工学研究科   生体工学専攻修士課程
• 1987年04月 - 1991年03月   北海道大学   工学部   電子工学科

所属学協会

• American Society for Biochemistry and Molecular Biology   日本蛋白質科学会   日本生化学会   日本生物物理学会   The Japanease Biochemical Society   The Biophysical Society of Japan   

研究活動情報

論文

• Configurational Changes of Retinal Schiff Base during Membrane Na+ Transport by a Sodium Pumping Rhodopsin.

  Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno

  The journal of physical chemistry letters 15 7 1993 - 1998 2024年02月22日 [査読有り][通常論文]
   

  Microbial rhodopsins are photoreceptors containing the retinal Schiff base chromophore and are ubiquitous among microorganisms. The Schiff base configuration of the chromophore, 15-anti (C═N trans) or 15-syn (C═N cis), is structurally important for their functions, such as membrane ion transport, because this configuration dictates the orientation of the positively charged NH group that interacts with substrate ions. The 15-anti/syn configuration is thus essential for elucidating the ion-transport mechanisms in microbial rhodopsins. Here, we identified the Schiff base configuration during the photoreaction of a sodium pumping rhodopsin from Indibacter alkaliphilus using Raman spectroscopy. We found that the unique configurational change from the 13-cis, 15-anti to all-trans, 15-syn form occurs between the photointermediates termed O1 and O2, which accomplish the Na+ uptake and release, respectively. This isomerization is considered to give rise to the highly irreversible O1 → O2 step that is crucial for unidirectional Na+ transport.
• Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

  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][通常論文]
   

  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.
• The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1 is enhanced by its extended cytoplasmic domain.

  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.
• A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites

  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.
• Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

  Risa Matsunami-Nakamura, Jun Tamogami, Miki Takeguchi, Junya Ishikawa, Takashi Kikukawa, Naoki Kamo, Toshifumi Nara

  FEBS letters 2023年08月02日 [査読有り]
   

  The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.
• Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

  Yi Wang, Yuchi Song, Shaonan Yan, Rina Hiramine, Yuki Ohnishi, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Antibiotics 12 6 1047 - 1047 2023年06月14日 [査読有り]
   

  Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.
• Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.

  Akiko Omori, Shotaro Sasaki, Takashi Kikukawa, Kazumi Shimono, Seiji Miyauchi

  Biochemistry 62 11 1608 - 1618 2023年06月06日 [査読有り]
   

  Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.
• Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity.

  Masaiku Ohya, Takashi Kikukawa, Junpei Matsuo, Takashi Tsukamoto, Ryota Nagaura, Tomotsumi Fujisawa, Masashi Unno

  The journal of physical chemistry. B 2023年05月18日 [査読有り]
   

  Chloride transport by microbial rhodopsins is actively being researched to understand how light energy is converted to drive ion pumping across cell membranes. Chloride pumps have been identified in archaea and eubacteria, and there are similarities and differences in the active site structures between these groups. Thus, it has not been clarified whether a common mechanism underlies the ion pump processes for all chloride-pumping rhodopsins. Here, we applied Raman optical activity (ROA) spectroscopy to two chloride pumps, Nonlabens marinus rhodopsin-3 (NM-R3) and halorhodopsin from the cyanobacterium Mastigocladopsis repens (MrHR). ROA is a vibrational spectroscopy that provides chiral sensitivity, and the sign of ROA signals can reveal twisting of cofactor molecules within proteins. Our ROA analysis revealed that the retinal Schiff base NH group orients toward the C helix and forms a direct hydrogen bond with a nearby chloride ion in NM-R3. In contrast, MrHR is suggested to contain two retinal conformations twisted in opposite directions; one conformation has a hydrogen bond with a chloride ion like NM-R3, while the other forms a hydrogen bond with a water molecule anchored by a G helix residue. These results suggest a general pump mechanism in which the chloride ion is "dragged" by the flipping Schiff base NH group upon photoisomerization.
• Phenylazothiazoles as Visible-Light Photoswitches

  Runze Lin, P. K. Hashim, Saugata Sahu, Ammathnadu S. Amrutha, Nusaiba Madappuram Cheruthu, Shakkeeb Thazhathethil, Kiyonori Takahashi, Takayoshi Nakamura, Takashi Kikukawa, Nobuyuki Tamaoki

  Journal of the American Chemical Society 2023年04月12日 [査読有り]
  
• Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

  Yuchi Song, Yi Wang, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Microbial cell factories 22 1 9 - 9 2023年01月13日 [査読有り]
   

  BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.
• Mutations conferring SO42- pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant.

  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.
• Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues

  Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa

  ACS Omega 7 36 31924 - 31934 2022年09月02日 [査読有り]
  
• Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin.

  Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno

  The Journal of biological chemistry 298 9 102366 - 102366 2022年09月 [査読有り]
   

  Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the "O intermediate", performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na+ are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na+ transport mediated by the chromophore-binding pocket of NaRs.
• Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

  Yuji Sato, Yi Wang, Yuchi Song, Weiming Geng, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Makoto Demura, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Amino acids 54 289 - 297 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.
• Pore Size Distributions Related with Spontaneous Purple Membrane Stacking in Porous Hydrogels

  Morise Karasawa, Yasunori Yokoyama, Kingo Takiguchi, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka

  Journal of the Physical Society of Japan 90 10 103801 - 103801 2021年10月15日 [査読有り][通常論文]
  
• Comparison of functionality and structural stability of bacteriorhodopsin reconstituted in partially fluorinated dimyristoylphosphatidylcholine liposomes with different perfluoroalkyl chain lengths.

  Mami Hashimoto, Yuka Murai, Kohei Morita, Takashi Kikukawa, Toshiyuki Takagi, Hiroshi Takahashi, Yasunori Yokoyama, Hideki Amii, Masashi Sonoyama

  Biochimica et biophysica acta. Biomembranes 1863 10 183686 - 183686 2021年10月01日 [査読有り][通常論文]
   

  Amphiphilic molecules with one or more perfluoroalkyl groups (Rf, CnF2n+1), which show peculiar interfacial properties, are attracting much attention in membrane protein science. We recently have developed a partially fluorinated dimyristoylphosphatidylcholine (DMPC) with a perfluorobutyl group in the hydrophobic chain terminal (F4-DMPC) and demonstrated that F4-DMPC is a promising material for incorporating membrane proteins. Moreover, we have found out that membrane properties of a series of partially fluorinated DMPCs with different Rf chain lengths (Fn-DMPCs) vary in a significant Rf chain length-dependent manner. In the present study, structural and functional properties of a membrane protein bacteriorhodopsin (bR) in the Fn-DMPC (n = 4, 6, and 8) membranes (bR/Fn-DMPC) are investigated using several physicochemical techniques. Regardless of the Rf chain lengths, bR/Fn-DMPCs retain native-like structural and functional properties at 30 °C, unlike bR molecules in DMPC vesicles. In particular, bR/F6-DMPC, which is in the fluid phase at 30 °C, shows flash-induced transient absorption changes very similar to the native purple membrane (PM) and very high thermal stability of bR trimers comparable to the PM. Structural and functional properties of bR/Fn-DMPCs are discussed compared to the PM and bR/DMPC.
• Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins.

  Syogo Sasaki, Jun Tamogami, Koki Nishiya, Makoto Demura, Takashi Kikukawa

  The Journal of biological chemistry 297 3 101013 - 101013 2021年07月28日 [査読有り][通常論文]
   

  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.
• Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization.

  Masuzu Kikuchi, Keiichi Kojima, Shin Nakao, Susumu Yoshizawa, Shiho Kawanishi, Atsushi Shibukawa, Takashi Kikukawa, Yuki Sudo

  Scientific reports 11 1 14765 - 14765 2021年07月20日 [査読有り][通常論文]
   

  Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.
• Preference of Proteomonas sulcata anion channelrhodopsin for NO3- revealed using a pH electrode method.

  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][通常論文]
   

  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.
• Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: insight of Na+ binding for active Na+ transport.

  Yushi Nakamizo, Tomotsumi Fujisawa, Takashi Kikukawa, Akiko Okamura, Hiroaki Baba, Masashi Unno

  Physical chemistry chemical physics : PCCP 23 3 2072 - 2079 2021年01月28日 [査読有り][通常論文]
   

  We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.
• Cl^–ポンプロドプシンの分子機構とH⁺ポンプへの機能変換

  菊川 峰志

  生物物理 61 1 012 - 015 一般社団法人 日本生物物理学会 2021年 [査読有り][招待有り]
   

  Cl^–-pump rhodopsin is the second discovered microbial rhodopsin and functions as light-driven Cl^– pump. The physiological significance of the Cl^– pump has not been fully resolved. However, its functional mechanism has been studied as a model system of anion transporters. In this review, the variation and functional mechanisms of Cl^–-pump rhodopsins were summarized. After 2014, novel Cl^–-pump groups were discovered in marine and terrestrial bacteria and were revealed to have unique characteristics. The most recently identified protein has close similarity with the H⁺-pump rhodopsin and begins to pump H⁺ outwardly by only a single amino acid replacement.
• Unique Cl^– pump rhodopsin with close similarity to H⁺ pump rhodopsin

  Takashi Kikukawa

  Biophysics and Physicobiology 18 317 - 326 2021年 [査読有り][招待有り]
  
• Spectroscopic approach for exploring structure and function of photoreceptor proteins.

  Masashi Unno, Yuu Hirose, Masaki Mishima, Takashi Kikukawa, Tomotsumi Fujisawa, Tatsuya Iwata, Jun Tamogami

  Biophysics and physicobiology 18 127 - 130 2021年 [査読有り][招待有り]
  
• Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity.

  Akiko Omori, Yuki Fujisawa, Shotaro Sasaki, Kazumi Shimono, Takashi Kikukawa, Seiji Miyauchi

  Biological & pharmaceutical bulletin 44 5 678 - 685 2021年 [査読有り][通常論文]
   

  To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [3H]-Gly-Sar. The efflux of [3H]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [3H]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbälch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pKa value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.
• Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin

  Tomoya Kato, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa

  Journal of Biological Chemistry 296 100792 - 100792 2021年01月 [査読有り][通常論文]
   

  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.
• Effects of salt and gel network structures on purple membrane stacking in hydrogels immobilized with poly(vinyl alcohol)

  Yasunori Yokoyama, Shunsuke Yano, Riku Kurita, Morise Karasawa, Hikaru Tanaka, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka

  JOURNAL OF APPLIED PHYSICS 129 1 2021年01月 [査読有り][通常論文]
   

  The effects of a gel network structure on purple membrane (PM) spontaneous stacking are discussed, with particular emphasis on the relationship between the PM periodic pre-structures before gelation. Such peculiar intermembrane pre-structures with a periodic distance of similar to 20nm have recently been found to provide an important key to PM stacking, which is critical for the engineering applications of PM. Here, we investigated how the PM stacking differs depending on the gel network of a poly(vinyl alcohol) gel. On the basis of the results, we propose a plausible interplay between the PM periodic pre-structures and the type of gel network for PM stacking. The results indicate that a porous gel network, in addition to the PM periodic pre-structure in solutions, is necessary for PM stacking in immobilized gel samples.
• Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift.

  Keiichi Kojima, Susumu Yoshizawa, Masumi Hasegawa, Masaki Nakama, Marie Kurihara, Takashi Kikukawa, Yuki Sudo

  Scientific reports 10 1 20857 - 20857 2020年11月30日 [査読有り][通常論文]
   

  The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named 'schizorhodopsin (SzR)' has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral 'blue-shift' due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.
• "Watching" a Molecular Twist in a Protein by Raman Optical Activity.

  Junpei Matsuo, Takashi Kikukawa, Tomotsumi Fujisawa, Wouter D Hoff, Masashi Unno

  The journal of physical chemistry letters 11 20 8579 - 8584 2020年09月25日 [査読有り][通常論文]
   

  Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.
• Direct Detection of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin.

  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.
• Photoisomerization of azobenzene units drives the photochemical reaction cycles of proteorhodopsin and bacteriorhodopsin analogues.

  Shariful Haque, Takashi Kikukawa, Nobuyuki Tamaoki

  Organic & biomolecular chemistry 18 32 6312 - 6327 2020年08月19日 [査読有り][通常論文]
   

  In this study we substituted the retinal units in proteorhodopsin (PR) and bacteriorhodopsin (BR) with azo chromophores to investigate the mechanism of photoinduced proton pumping in rhodopsins and potentially develop new artificial molecular pumps. We used an indium tin oxide electrode to investigate the photoinduced proton transfer of the azo analogues of PR and BR. We also employed flash photolysis to determine the characteristic photocycles, comprising multiple transient intermediates, of the azo chromophore-bound PR and BR. Moreover, our studies of the photoinduced proton pumping functions of azo-proteoopsin and azo-bacterioopsin complexes revealed that they did not pump protons upon illumination, even though they underwent photoinduced proton transfer and the characteristic photocycle. Mutational analysis suggested that the proton pumping malfunction of the azo analogues of PR and BR resulted from the absence of proton transfer reactions through cytoplasmic channels, even though these reactions were evoked in extracellular channels. Based on our experimental findings, we propose herein a putative model of the proton transfer reaction mechanism for the azo analogues of PR and BR.
• Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

  Sato T, Kikukawa T, Miyoshi R, Kajimoto K, Yonekawa C, Fujisawa T, Unno M, Eki T, Hirose Y

  The Journal of biological chemistry 294 49 18909 - 18922 2019年10月 [査読有り][通常論文]
   

  Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state ( 15Z Pb) and orange-absorbing 15E state ( 15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( 15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to- 15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.
• Low-Temperature Raman Spectroscopy of Halorhodopsin from Natronomonas pharaonis: Structural Discrimination of Blue-Shifted and Red-Shifted Photoproducts.

  Fujisawa T, Kiyota H, Kikukawa T, Unno M

  Biochemistry 58 40 4159 - 4167 2019年10月 [査読有り][通常論文]
   

  From the low-temperature absorption and Raman measurements of halorhodopsin from Natronomonas pharaonis (pHR), we observed that the two photoproducts were generated after exciting pHR at 80 K by green light. One photoproduct was the red-shifted K intermediate (pHR(K)) as the primary photointermediate for Cl- pumping, and the other was the blue-shifted one (pHR(hypso)), which was not involved in the Cl- pumping and thermally relaxed to the original unphotolyzed state by increasing temperature. The formation of these two kinds of photoproducts was previously reported for halorhodopsin from Halobacterium sarinarum [Zimanyi et al. Bio-K chemistry 1989, 28, 1656]. We found that the same took place in pHR, and we revealed the chromophore structures of the two photo intermediates from their Raman spectra for the first time. pHR(hypso) had the distorted all-trans chromophore, while pHR(K) contained the distorted 13-cis form. The present results revealed that the structural analyses of pHR(K) carried out so far at similar to 80 K potentially included a significant contribution from pHR(hypso). pHR(hypso) was efficiently formed via the photoexcitation of pHR(K), indicating that pHR(hypso) was likely a side product after photoexcitation of pHR(K). The formation of pHR(hypso) suggested that the active site became tight in pHR(K) due to the slight movement of Cl-, and the back photoisomerization then produced the distorted all-trans chromophore in pHR(hypso).
• Functional importance of the oligomer formation of the cyanobacterial H⁺ pump Gloeobacter rhodopsin.

  Iizuka A, Kajimoto K, Fujisawa T, Tsukamoto T, Aizawa T, Kamo N, Jung KH, Unno M, Demura M, Kikukawa T

  Scientific reports 9 1 10711  2019年07月 [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.
• Comparison of two different partially fluorinated phosphatidylcholines with the perfluorobutyl group on thermotropic properties of the bilayer membrane and reconstituted bacteriorhodopsin.

  Yanagi T, Takagi T, Takahashi H, Kikukawa T, Amii H, Sonoyama M

  Biophysical reviews 11 3 395 - 398 2019年05月 [査読有り][通常論文]
  
• Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.

  Takahashi H, Yoshino M, Morita K, Takagi T, Yokoyama Y, Kikukawa T, Amii H, Kanamori T, Sonoyama M

  Biochimica et biophysica acta. Biomembranes 1861 3 631 - 642 2019年03月 [査読有り][通常論文]
   

  This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.
• Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.

  Hasemi T, Kikukawa T, Watanabe Y, Aizawa T, Miyauchi S, Kamo N, Demura M

  Biochimica et biophysica acta. Bioenergetics 1860 2 136 - 146 2019年02月 [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.
• Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle.

  Geng X, Dai G, Chao L, Wen D, Kikukawa T, Iwasa T

  Photochemistry and photobiology 95 4 980 - 989 2018年12月 [査読有り][通常論文]
  
• Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl^- concentrations.

  Tsukamoto T, Kikuchi C, Suzuki H, Aizawa T, Kikukawa T, Demura M

  Scientific reports 8 1 13445 - 13445 2018年09月 [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-.
• Low-temperature Raman spectroscopy reveals small chromophore distortion in primary photointermediate of proteorhodopsin.

  Fujisawa T, Abe M, Tamogami J, Kikukawa T, Kamo N, Unno M

  FEBS letters 592 18 3054 - 3061 2018年08月 [査読有り][通常論文]
   

  Proteorhodopsin (PR) is a microbial rhodopsin functioning as a light-driven proton pump in aquatic bacteria. We performed low-temperature Raman measurements of PR to obtain the structure of the primary photoproduct, the K intermediate (PRK). PRK showed the hydrogen-out-of-plane modes that are much less intense than those of bacteriorhodopsin as the prototypical light-driven proton pump from haloarchaea. The present results reveal the significantly relaxed chromophore structure in PRK, which can be coupled to the slow kinetics of the K intermediate. This structure suggests that PR transports protons using the small energy storage within the chromophore at the start of its photocycle.
• Interhelical interactions between D92 and C218 in the cytoplasmic domain regulate proton uptake upon N-decay in the proton transport of Acetabularia rhodopsin II

  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.
• Presence of a Haloarchaeal Halorhodopsin-Like Cl- Pump in Marine Bacteria.

  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.
• Photocycle of Sensory Rhodopsin II from Halobacterium salinarum (HsSRII): Mutation of D103 Accelerates M Decay and Changes the Decay Pathway of a 13-cis O-like Species.

  Dai G, Geng X, Chao L, Tamogami J, Kikukawa T, Demura M, Kamo N, Iwasa T

  Photochemistry and photobiology 94 4 705 - 714 2018年03月 [査読有り][通常論文]
  
• Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.

  Saki Inoue, Susumu Yoshizawa, Yu Nakajima, Keiichi Kojima, Takashi Tsukamoto, Takashi Kikukawa, Yuki Sudo

  Physical chemistry chemical physics : PCCP 20 5 3172 - 3183 2018年01月31日 [査読有り][通常論文]
   

  A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.
• Targeted Activation of Molecular Transportation by Visible Light

  Ammathnadu S. Amrutha, K. R. Sunil Kumar, Takashi Kikukawa, Nobuyuki Tamaoki

  ACS Nano 11 12 12292 - 12301 2017年12月26日 [査読有り][通常論文]
   

  Regulated transportation of nanoscale objects with a high degree of spatiotemporal precision is a prerequisite for the development of targeted molecular delivery. In vitro integration of the kinesin-microtubule motor system with synthetic molecules offers opportunities to develop controllable molecular shuttles for lab-on-a-chip applications. We attempted a combination of the kinesin-microtubule motor system with push-pull type azobenzene tethered inhibitory peptides (azo-peptides) through which reversible, spatiotemporal control over the kinesin motor activity was achieved locally by a single, visible wavelength. The fast thermal relaxation of the cis-isomers of azo-peptides offered us quick and complete resetting of the trans-state in the dark, circumventing the requirement of two distinct wavelengths for two-way switching of kinesin-driven microtubule motility. Herein, we report the manipulation of selected, single microtubule movement while keeping other microtubules at complete rest. The photoresponsive inhibitors discussed herein would help in realizing complex bionanodevices.
• Enhanced expression of cysteine-rich antimicrobial peptide snakin-1 in Escherichia coli using an aggregation-prone protein coexpression system

  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
• Spontaneous stacking of purple membranes during immobilization with physical cross-linked poly(vinyl alcohol) hydrogel with retaining native-like functionality of bacteriorhodopsin

  Yasunori Yokoyama, Hikaru Tanaka, Shunsuke Yano, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka

  JOURNAL OF APPLIED PHYSICS 121 20 2017年05月 [査読有り][通常論文]
   

  We previously discovered the correlation between light-induced chromophore color change of a photo-receptor membrane protein bacteriorhodopsin (bR) and its two-dimensional crystalline state in the membrane. To apply this phenomenon to a novel optical memory device, it is necessary that bR molecules are immobilized as maintaining their structure and functional properties. In this work, a poly(vinyl alcohol) (PVA) hydrogel with physical cross-linkages (hydrogen bonds between PVA chains) that resulted from repeated freezing-and-thawing (FT) cycles was used as an immobilization medium. To investigate the effects of physically cross-linked PVA gelation on the structure and function of bR in purple membranes (PMs), spectroscopic techniques were employed against PM/PVA immobilized samples prepared with different FT cycle numbers. Visible circular dichroism spectroscopy strongly suggested PM stacking during gelation. X-ray diffraction data also indicated the PM stacking as well as its native-like crystalline lattice even after gelation. Timeresolved absorption spectroscopy showed that bR photocycle behaviors in PM/PVA immobilized samples were almost identical to that in suspension. These results suggested that a physically crosslinked PVA hydrogel is appropriate for immobilizing membrane proteins in terms of maintaining their structure and functionality. Published by AIP Publishing.
• Transient Resonance Raman Spectroscopy of a Light-Driven Sodium-Ion-Pump Rhodopsin from Indibacter alkaliphilus

  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.
• Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics

  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.
• Existence of two O-like intermediates in the photocycle of Acetabularia rhodopsin II, a light-driven proton pump from a marine alga

  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.
• Photochemical characterization of actinorhodopsin and its functional existence in the natural host

  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.
• Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

  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.
• Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

  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.
• In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor

  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.
• Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

  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.
• Structure determination of uniformly C-13, N-15 labeled protein using qualitative distance restraints from MAS solid-state C-13-NMR observed paramagnetic relaxation enhancement

  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.
• Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump

  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.
• Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum

  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.
• Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

  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.
• Probing the Cl--pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

  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.
• Microbial rhodopsins of Halorubrum species isolated from Ejinoor salt lake in Inner Mongolia of China

  Chaoluomeng, Gang Dai, Takashi Kikukawa, Kunio Ihara, Tatsuo Iwasa

  PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES 14 11 1974 - 1982 2015年 [査読有り][通常論文]
   

  Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.
• The effects of chloride ion binding on the photochemical properties of sensory rhodopsin II from Natronomonas pharaonis

  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.
• Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide.

  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 2014年03月 [査読有り][通常論文]
  
• Role of Thr218 in the Light-Driven Anion Pump Halorhodopsin from Natronomonas pharaonis

  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.
• Physicochemical Studies of Bacteriorhodopsin Reconstituted in Partially Fluorinated Phosphatidylcholine Bilayers

  Masaru Yoshino, Takashi Kikukawa, Hiroshi Takahashi, Toshiyuki Takagi, Yasunori Yokoyama, Hideki Amii, Teruhiko Baba, Toshiyuki Kanamori, Masashi Sonoyama

  JOURNAL OF PHYSICAL CHEMISTRY B 117 18 5422 - 5429 2013年05月 [査読有り][通常論文]
   

  A membrane protein bacteriorhodopsin (bR) that is successfully reconstituted in liposome of a novel partially fluorinated analog of dimyristoylphosphatidylcholine (DMPC) with the perfluorobutyl segments in the myristoyl groups, diF4H10-PC, has been investigated by some spectroscopic and X-ray diffraction techniques to clarify effects of substitution of nine hydrogen atoms by fluorine atoms on structural and physical properties of the membrane protein by comparison with the previous results on proteoliposome of bR and DMPC. Below the gel-to-liquid crystalline phase transition of diF4H10-PC bilayer, bR molecules adopt the two-dimensional lattice structure of trimers as the structural unit and show a photocycle very similar to that of native purple membrane like reconstituted bR in DMPC liposome in the gel phase. Even upon heating up to temperatures well above the phase transition, the nativelike functional reconstitution and higher structural stability of bR molecules in diF4H10-PC liposome are retained, which strikingly contrasts with lipid phase transition-induced disaggregation of protein molecules and light-induced denaturation in DMPC liposome. Greater membrane rigidity and low affinity between bR and fluorinated lipid molecules are proposed as a driving force for keeping nativelike properties of bR molecules in diF4H10-PC liposome even in the fluid phase.
• Salt bridge in the conserved His-Asp cluster in Gloeobacter rhodopsin contributes to trimer formation

  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.
• Exploring the Active Site Structure of a Photoreceptor Protein by Raman Optical Activity

  Masashi Unno, Takashi Kikukawa, Masato Kumauchi, Naoki Kamo

  JOURNAL OF PHYSICAL CHEMISTRY B 117 5 1321 - 1325 2013年02月 [査読有り][通常論文]
   

  We have developed a near-infrared excited Raman optical activity (ROA) spectrometer and report the first measurement of near-infrared ROA spectra of a light-driven proton pump, bacteriorhodopsin. Our results demonstrate that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, the ROA spectra of the all-trans, 15-anti and 13-cis, 15-syn isomers differ significantly, indicating a high structural sensitivity of the ROA spectra. We therefore expect that future applications of the near-infrared ROA will allow the experimental elucidation of the active site structures in other proteins as well as reaction intermediates.
• Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry

  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.
• Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

  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 - 8 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.
• A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method

  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.
• Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of Salinibacter Sensory Rhodopsin I

  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.
• Photoinduced Proton Release in Proteorhodopsin at Low pH: The Possibility of a Decrease in the pK(a) of Asp227

  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.
• Dynamics of Dangling Bonds of Water Molecules in pharaonis Halorhodopsin during Chloride Ion Transportation

  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
• Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

  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.
• Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacterium salinarum by hydroxylamine: Identification of the responsible intermediates

  Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Makoto Demura, Toshifumi Nara, Naoki Kamo

  JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 106 1 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]
  
• Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state.

  Yamashita Y, Kikukawa T, Tsukamoto T, Kamiya M, Aizawa T, Kawano K, Miyauchi S, Kamo N, Demura M

  Biochimica et biophysica acta 1808 12 2905 - 2912 2011年12月 [査読有り][通常論文]
  
• Photochemistry of Acetabularia Rhodopsin II from a Marine Plant, Acetabularia acetabulum

  Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Seiji Miyauchi, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Kwang-Hwan Jung, Shigeyuki Yokoyama, Naoki Kamo

  BIOCHEMISTRY 50 41 8888 - 8898 2011年10月 [査読有り][通常論文]
   

  Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARE is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARE was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P,) were sufficient to simulate the data. Noticeably, both P-3 and P-4 contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (G1u204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.
• Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga

  Takashi Wada, Kazumi Shimono, Takashi Kikukawa, Masakatsu Hato, Naoko Shinya, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Jun Tamogami, Seiji Miyauchi, Kwang-Hwan Jung, Naoki Kamo, Shigeyuki Yokoyama

  JOURNAL OF MOLECULAR BIOLOGY 411 5 986 - 998 2011年09月 [査読有り][通常論文]
   

  Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 A resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARM is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the "late proton release". (C) 2011 Elsevier Ltd. All rights reserved.
• Spectroscopic Evidence for the Formation of an N Intermediate during the Photocycle of Sensory Rhodopsin II (Phoborhodopsin) from Natronobacterium pharaonis

  Yusuke Tateishi, Takayuki Abe, Jun Tamogami, Yutaka Nakao, Takashi Kikukawa, Naoki Kamo, Masashi Unno

  BIOCHEMISTRY 50 12 2135 - 2143 2011年03月 [査読有り][通常論文]
   

  Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a fight-driven proton pump, the photocycle is established as BR -> K -> L -> M -> N -> O -> BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the 0, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is similar to 500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.
• A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-like Properties

  Yuki Sudo, Kunio Ihara, Shiori Kobayashi, Daisuke Suzuki, Hiroki Irieda, Takashi Kikukawa, Hideki Kandori, Michio Homma

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 8 5967 - 5976 2011年02月 [査読有り][通常論文]
   

  Rhodopsins possess retinal chromophore surrounded by seven transmembrane alpha-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photo-receptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.
• Photochemistry of a putative new class of sensory rhodopsin (SRIII) coded by xop2 of Haloarcular marismortui

  Yutaka Nakao, Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Noriko Kimitsuki, Toshifumi Nara, Masashi Unno, Kunio Ihara, Naoki Kamo

  JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 102 1 45 - 54 2011年01月 [査読有り][通常論文]
   

  Baliga et al. (2004) [1] reported the existence of a functionally unpredictable opsin gene, named xop2, in Haloarcula marismortui, a holophilic archaeon. Ihara et al. [38] performed molecular phylogenetic analysis and determined that the product of xop2 belonged to a new class of opsins in the sensory rhodopsins. This microbial rhodopsin was therefore named H. marismortui sensory rhodopsin III (HmSRIII). Here, we functionally expressed HmSRIII in Escherichia coli cell membranes to examine the photochemistry. The wavelength of maximum absorption (lambda(max)) for HmSRIII was 506 nm. We observed a very slow photocycle that completed in similar to 50 s. Intermediates were defined as M (lambda(max) similar to 380 nm), N (lambda(max) similar to 460 nm) and O (lambda(max) similar to 530 nm) 0.01 s after the flash excitation. The nomenclature for these intermediates was based on their locations along the absorption maxima of bacteriorhodopsin. Analysis of laser-flash-photolysis data in the presence and absence of azide gave the following results: (1) an equilibrium between N and 0 was attained, (2) the direct product of the M-decay was 0 but not N, and (3) the last photo-intermediate (HmSRIII') had a lambda(max) similar to that of the original, and its decay rate was very slow. Resonance Raman spectroscopy revealed that this N-intermediate had 13-cis retinal conformation. Proton uptake occurred during the course of M-decay, whereas proton release occurred during the course of O-decay (or exactly N-O equilibrium). Very weak proton-pumping activity was observed whose direction is the same as that of bacteriorhodopsin, a typical light-driven proton pump. (C) 2010 Elsevier B.V. All rights reserved.
• ＳｎＯ２透明電極を用いた光受容タンパク質のプロトン移動解析

  田母神 淳, 菊川 峰志

  生物物理 51 1 042 - 043 一般社団法人 日本生物物理学会 2011年 [査読有り]
  
• STPR, a 23-Amino Acid Tandem Repeat Domain, Found in the Human Function-Unknown Protein ZNF821

  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.
• Photoreaction Cycle of Phoborhodopsin (Sensory Rhodopsin II) from Halobacterium salinarum Expressed in Escherichia coli

  Gang Dai, Yoshikazu Ohno, Yoichi Ikeda, Jun Tamogami, Takashi Kikukawa, Naoki Kamo, Tatsuo Iwasa

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 86 3 571 - 579 2010年05月 [査読有り][通常論文]
   

  Phoborhodopsin (pR; also called sensory rhodopsin II, SRII) is a photoreceptor of negative phototaxis of halobacteria. The studies of photochemical properties of this pigment are not many because the amount of the pigment is small and the stability is low. Recently an expression system of phoborhodopsin from Halobacterium salinarum (called salinarum phoborhodopsin, spR; also HsSRII) in Escherichia coli and purification method has been developed (Mironova et al. [2005] FEBS Lett., 579, 3147-3151), which enables detailed studies on the photochemical properties of spR. In the present work, the photoreaction cycle of E. coli-expressed spR was studied by low-temperature spectroscopy and flash photolysis. Formations of K-, M-, O-like intermediates and P480 were reconfirmed as reported previously. New findings are as follows. (1) The K-like intermediate (P500) was a mixture of two photoproducts. (2) Formation of L-like intermediate (P482) was observed by low-temperature spectroscopy and flash photolysis at room temperature. (3) On long irradiation of spR at 20 degrees C, formation of a new photoproduct P370 was observed and it decayed to the original spR in the dark with a decay half time of 190 min. Based on these results the similarities and dissimilarities between spR and ppR are discussed.
• The Photochemical Reaction Cycle and Photoinduced Proton Transfer of Sensory Rhodopsin II (Phoborhodopsin) from Halobacterium salinarum

  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.
• Kinetic study of anti-viral ribavirin uptake mediated by hCNT3 and hENT1 in Xenopus laevis oocytes

  Takashi Yamamoto, Mitsuru Sugawara, Takashi Kikukawa, Seiji Miyauchi, Masahiro Yamaguchi, Atsushi Tero, Seiji Takagi, Toshiyuki Nakagaki

  BIOPHYSICAL CHEMISTRY 147 1-2 59 - 65 2010年03月 [査読有り][通常論文]
   

  Transport across the cell membrane is crucial in drug delivery. However, the process is complicated because nucleoside derivatives that are commonly used its anti-viral drugs are transported through two different types of specific transporters: concentrative transporters and equilibrative transporters. Cross-disciplinary approaches involving both biological experiments and theoretical considerations are therefore necessary to study the transport of nucleoside analogues such as ribavirin. Here we constructed an experimental model system using the Xenopus laevis oocyte that expressed examples of both types of transporters: human concentrative nucleoside transporter 3 and human equilibrative transporter 1. We also performed a kinetic study. Experimental results showed that the transport of ribavirin could be reduced by inhibiting one of the two types of transporters, which seems to be counterintuitive. We therefore designed a simple mathematical model of the dynamics of ribavirin uptake and analyzed the model behaviors using a numerical simulation. The theoretical results reproduced the experimentally observed phenomena and suggested a possible mechanism for the process. Based on this mechanism, we predicted some potential methods for the effective uptake of ribavirin from a dynamics point of view. (C) 2010 Elsevier B.V. All rights reserved.
• Spectroscopic Studies of a Sensory Rhodopsin I Homologue from the Archaeon Haloarcula vallismortis

  Jin Yagasaki, Daisuke Suzuki, Kunio Ihara, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Michio Homma, Hideki Kandori, Yuki Sudo

  BIOCHEMISTRY 49 6 1183 - 1190 2010年02月 [査読有り][通常論文]
   

  Sensory rhodopsin 1 (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new Information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (Hi,SRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in Hi,SRI (unlike in SrSRI), and Hi,SRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of Hi,SRI under acidic and basic conditions and observed the effects of Cl- oil the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving Our understanding of the molecular mechanism of the dual photosensing by SRI.
• Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I (vol 392, pg 48, 2009)

  Suzuki, Daisuke, Furutani, Yuji, Inoue, Keiichi, Kikukawa, Takashi, Sakai, Makoto, Fujii, Masaaki, K, ori, Hideki, Homma, Michio, Sudo, Yuki

  Journal of Molecular Biology 395 1 2010年
• Effect of Molecular Assembly on Photocycle of Reconstituted Bacteriorhodopsin: Significant Blue Shift of the Late M Photointermediate in the Liquid Crystalline Phase

  Masashi Sonoyama, Takashi Kikukawa, Yasunori Yokoyama, Makoto Demura, Naoki Kamo, Shigeki Mitaku

  CHEMISTRY LETTERS 38 12 1134 - 1135 2009年12月 [査読有り][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.
• Effect of Chloride Binding on the Thermal Trimer-Monomer Conversion of Halorhodopsin in the Solubilized System

  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.
• Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid

  Kazumi Shimono, Mie Goto, Takashi Kikukawa, Seiji Miyauchi, Mikako Shirouzu, Naoki Kamo, Shigeyuki Yokoyama

  PROTEIN SCIENCE 18 10 2160 - 2171 2009年10月 [査読有り][通常論文]
   

  Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.
• Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I

  Daisuke Suzuki, Yuji Furutan, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Hideki Kandori, Michio Homma, Yuki Sudo

  JOURNAL OF MOLECULAR BIOLOGY 392 1 48 - 62 2009年09月 [査読有り][通常論文]
   

  Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl(-)-dependent manner with a K(m) of 307 +/- 56 mM, showing that Cl(-)-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO(3)) implying that I(-), Br(-), and NO(3)(-) can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl(-)-binding site. In light of these results, we discuss the effects of the Cl(-) binding to SRI and the roles of Cl(-) binding in its function. (C) 2009 Elsevier Ltd. All rights reserved.
• X-ray crystallography and structural stability of digestive lysozyme from cow stomach

  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.
• A Tin Oxide Transparent Electrode Provides the Means for Rapid Time-resolved pH Measurements: Application to Photoinduced Proton Transfer of Bacteriorhodopsin and Proteorhodopsin

  Jun Tamogami, Takashi Kikukawa, Seiji Miyauchi, Eiro Muneyuki, Naoki Kamo

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 2 578 - 589 2009年03月 [査読有り][通常論文]
   

  An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO(2) electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265: 762-765, 1994; Robertson and Lukashev, Biophys. J. 68: 1507-1517, 1995; Koyama et al., Photochem. Photobiol. 68: 400-406, 1998). In this paper, we concluded that pH changes caused by proton transfer by the deposited BR or proteorhodopsin (PR) films lead to the flash-induced potential change in the SnO2 electrode. Thus, the signals originate from BR and PR acting as light-driven proton pumps. This conclusion was drawn from the following observations. (1) The relation between the potential of a bare electrode and pH is linear for a wide pH range. (2) The flash-induced potential changes decrease with an increase in the buffer concentration. (3) The action spectrum of PR agrees well with the absorption spectrum. (4) The present electrode can monitor the pH change in the time range from 10 ms to several hundred milliseconds, as deduced by comparing the SnO2 signal with the signals of pH-sensitive dyes. Using this electrode system, flash-induced proton transfer by BR was measured for a wide pH range from 2 to 10. From these data, we reconfirmed various pK(a) values reported previously, indicating that the present method can give the correct pK(a) values. This is the first report to estimate these pK(a) values directly from the proton transfer. We then applied this method to flash-induced proton transfer of PR. We observed proton uptake followed by release for the pH range from 4 to 9.5, and in other pH ranges, proton release followed by uptake was observed.
• Role of Arg123 in Light-driven Anion Pump Mechanisms of pharaonis Halorhodopsin

  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.
• Halorhodopsin from Natronomonas pharaonis Forms a Trimer Even in the Presence of a Detergent, Dodecyl-beta-d-maltoside

  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.
• Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death

  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.
• The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers - Effects of hydrostatic pressure and stopped flow experiments

  Takashi Kikukawa, Chabita K. Saha, Sergei P. Balashov, Eleonora S. Imasheva, Dmitry Zaslavsky, Robert B. Gennis, Takayuki Abe, Naoki Kamo

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 84 4 880 - 888 2008年07月 [査読有り][通常論文]
   

  Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.
• Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE

  Toshifumi Nara, Tomoko Kouyama, Yuko Kurata, Takashi Kikukawa, Seiji Miyauchi, Naoki Kamo

  JOURNAL OF BIOCHEMISTRY 142 5 621 - 625 2007年11月 [査読有り][通常論文]
   

  EmrE in Escherichia coli belongs to the small multidrug resistance (SMR) transporter family. It functions as a homo-dimer, but the orientation of the two monomers in the membrane (membrane topology) is under debate. We expressed various single-cysteine EmrE mutants in E. coli cells lacking a major efflux transporter. Efflux from cells expressing the P55C or T56C mutant was blocked by the external application of membrane-impermeable SH-reagents. This is difficult to explain by the parallel topology configuration, because Pro55 and Thr56 are considered to be located in the cytoplasm. From both the periplasm and the cytoplasm, biotin-PE-maleimide, a bulky membrane-impermeable SH-reagent, could access the cysteine residue at the 25th position in the presence of transport substrates and at the 108th position. These observations support the anti-parallel topology in the membrane.
• Anti-parallel membrane topology of two components of EbrAB, a multidrug transporter

  Takashi Kikukawa, Seiji Miyauchi, Tsunehisa Araiso, Naoki Kamo, Toshifumi Nara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 358 4 1071 - 1075 2007年07月 [査読有り][通常論文]
   

  EbrAB is a multidrug-resistance transporter in Bacillus subtilis that belongs to the small multidrug resistance, and requires two polypeptides of both EbrA and EbrB, implying that it functions in the hetero-dimeric state. In this study, we investigated the transmembrane topologies of EbrA and EbrB. Various single-cysteine mutants were expressed in Escherichia coli cells, and the efflux activity was measured. Only mutants having a high activity were used for the topology experiments. The reactivity of a membrane impermeable NEM-fluorescein against the single cysteine of these fully functional mutants was examined when this reactive fluorophore was applied either from the outside or both sides of the cell membrane or in the denatured state. The results clearly showed that EbrA and EbrB have the opposite orientation within the membrane or an anti-parallel configuration. (C) 2007 Elsevier Inc. All rights reserved.
• Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven C- pump activity

  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 [査読有り][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.
• Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel

  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.
• Difference between Pharmacokinetics of Mycophenolic acid (MPA) in Rats and that in Humans is caused by Different affinities of MRP2 to a glucuronized form

  Yoh Takekuma, Haruka Kakiuchi, Koujiro Yamazaki, Seiji Miyauchi, Takashi Kikukawa, Naoki Kamo, Vadivel Ganapathy, Mitsuru Sugawara

  JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES 10 1 71 - 85 2007年01月 [査読有り][通常論文]
   

  PURPOSE. Mycophenolic acid ( MPA), an immunosuppressant, is excreted as its glucuronized form, MPAG. In humans, MPAG is mostly excreted into urine, whereas more than 80% of the dose is excreted into bile in rats. The aim of this study was to clarify the cause of the species difference. We investigated whether MPAG is a substrate of human organic anion transporters ( hOATs), and we compared the affinities of multi-drug resistance-associated protein 2 ( MRP2) for MPAG in rats and humans. METHODS. The inhibitory effects of MPAG on the uptake of typical substrates via hOAT1 and hOAT3 were determined using HeLa cells heterologously expressing hOAT1 and Xenopus laevis oocytes heterologously expressing hOAT3. MPAG transport activity via hOAT1 and hOAT3 was determined by the two-microelectrode voltage-clamp technique using Xenopus laevis oocytes expressing hOAT1 and hOAT3. The affinities of MPAG for hMRP2 and rMrp2 were determined by the inhibitory effects of MPAG on p-aminohippuric acid ( a typical substrate) uptake using membrane vesicles expressing hMRP2 or rMrp2. RESULTS. MPAG inhibited the uptake of PAH via hOAT1 and hOAT3, and calculated IC50 values were 222.6 +/- 26.6 mu M and 41.5 +\- 11.5 mu M, respectively. However, MPAG was not transported by hOAT1 and hOAT3. MPAG strongly inhibited the uptake of PAH via both rMrp2 and hMRP2. However, the magnitudes of inhibitory effects were different. The calculated IC50 values were 286.2 +/- 157.3 mu M and 1036.8 +/- 330.5 mu M, respectively. CONCLUSION. MPAG is not a substrate but is an inhibitor of hOAT1 and hOAT3. The affinity of rMRP2 to MPAG was about 3.6 times as high as that of hMRP2. Therefore, the difference of affinity between hMRP2 and rMrp2 is a possible mechanism of the difference of excretion ratio of MPAG between rats and human.
• ヘテロダイマー型ＳＭＲ：ＥｂｒＡＢの膜内トポロジーの決定とモノマー単独での機能発現

  菊川 峰志, 奈良 敏文

  生物物理 47 4 264 - 267 一般社団法人 日本生物物理学会 2007年 [査読有り][通常論文]
  
• All-optical switching in pharaonis phoborhodopsin protein molecules

  Sukhdev Roy, Takashi Kikukawa, Parag Sharma, Naoki Kamo

  IEEE TRANSACTIONS ON NANOBIOSCIENCE 5 3 178 - 187 2006年09月 [査読有り][通常論文]
   

  Low-power all-optical switching with pharaonis phoborhodopsin (ppR) protein is demonstrated based on nonlinear excited-state absorption at different wavelengths. A modulating pulsed 532-nm laser beam is shown to switch the transmission of a continuous-wave signal light beam at: 1) 390 nm; 2) 500 nm; 3) 560 nm; and 4) 600 run, respectively. Simulations based on the rate equation approach considering all seven states in the ppR photocycle are in good agreement with experimental results. It is shown that the switching characteristics at 560 and 600 nm, respectively, can exhibit negative to positive switching. The switching characteristics at 500 nm can be inverted by increasing the signal beam intensity. The profile of switched signal beam is also sensitive to the modulating pulse frequency and signal beam intensity and wavelength. The switching characteristics are also shown to be sensitive to the lifetimes of ppR(M) and ppR(O) intermediates. The results show the applicability of ppR as a low-power wavelength tunable all-optical switch.
• Two-component bacterial multidrug transporter, EbrAB: Mutations making each component solely functional

  Takashi Kikukawa, Toshifumi Nara, Tsunehisa Araiso, Seiji Miyauchi, Naoki Kamo

  BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1758 5 673 - 679 2006年05月 [査読有り][通常論文]
   

  EbrAB in Bacillus subtilis belongs to a novel small multidrug resistance (SMR) family of multidrug efflux pumps. EmrE in Escherichia coli, a representative of SMR, functions as a homo-oligomer in the membrane. On the other hand, EbrAB requires a hetero-oligomeric configuration consisting of two polypeptides, EbrA and EbrB. Although both polypeptides have a high sequence similarity, expression of either single polypeptide does not confer the multi drug-resistance. We performed mutation studies on EbrA and B to determine why EbrAB requires the hetero-oligomerization. Mutants of EbrA and B lacking both the hydrophilic loops and the C-terminus regions conferred the multi drug-resistance solely by each protein. This suggests that the hydrophilic loops and the C-terminus regions constrain them to their respective conformations upon the formation of the functional hetero-oligomer. (c) 2006 Elsevier B.V. All rights reserved.
• A light-driven proton pump from Haloterrigena turkmenica: Functional expression in Escherichia coli membrane and coupling with a H+ co-transporter

  N Kamo, T Hashiba, T Kikukawa, T Araiso, K Ihara, T Nara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 341 2 285 - 290 2006年03月 [査読有り][通常論文]
   

  A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H+ pump and was identified in a novel strain Halo terrigena sp. arg-4 (K. Ihara T. Uemura, l. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163-174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsin (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10-15 mg protein/L Culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination. (c) 2006 Elsevier Inc. All rights reserved.
• Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin

  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.
• Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

  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.
• Changes in lipid mobility associated with alamethicin incorporation into membranes

  T Kikukawa, T Araiso

  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 405 2 214 - 222 2002年09月 [査読有り][通常論文]
   

  The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a "wobbling" of the lipid hydrocarbon chain, These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration, (C) 2002 Elsevier Science (USA). All rights reserved.
• Pharmacological characterization of emotional responses to unpredictable microgravity conditions

  M Yoshioka, S Ohashi, T Kikukawa, T Araiso, S Yano, M Tamura

  JAPANESE JOURNAL OF PHARMACOLOGY 88 234P - 234P 2002年 [査読有り][通常論文]
  
• Effects of anxiolytics on hemodynamic responses to acute microgravity environment

  Yoshioka, M, Kikukawa, T, Ohashi, S, Araiso, T, Yano, S, Tamura, M

  Biological Sciences in Space 14 3 190 - 191 2000年10月 [査読有り][通常論文]
  
• Polymerization of pyrrole in microgravity

  T Nakamura, T Akutagawa, T Hasegawa, T Kikukawa, T Araiso, M Higuchi, K Hiratani

  SYNTHETIC METALS 101 1-3 78 - 79 1999年05月 [査読有り][通常論文]
   

  Oxidative polymerization of pyrrole derivatives in a microgravity environment was curried out using the JAMIC (Japan Microgravity Center) drop shaft where 10-seconds microgravity condition is available. The structural and electronic properties of the polymers were compared with those obtained in IG.
• Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin

  K Takao, T Kikukawa, T Araiso, N Kamo

  BIOPHYSICAL CHEMISTRY 73 1-2 145 - 153 1998年07月 [査読有り][通常論文]
   

  Natronobacterium pharaonis has retinal proteins, one of which is pharaonis phoborhodopsin, abbreviated as ppR (or called pharaonis sensory rhodopsin II, psR-II). This pigment protein functions as a photoreceptor of the negative phototaxis of this bacterium. On photoexcitation ppR undergoes photocycling; the photoexcited state relaxes in the dark and returns to the original state via several intermediates. The photocycle of ppR resembles that of bR except in wavelengths and rate. The cycle of bR is completed in 10 ms while that of ppR takes seconds. The Arrhenius analysis of M-intermediate (ppR(M)) decay which is rate-limiting revealed that the slow decay is due to the large negative activation entropy of ppR. The addition of azide increases the decay rate 300-fold (at pH 7); Arrhenius analysis revealed decreases in the activation energy (activation enthalpy) and a further decrease in the activation entropy. (C) 1998 Elsevier Science B.V. All rights reserved.
• Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol

  T Kikukawa, T Araiso, T Shimozawa, K Mukasa, N Kamo

  BIOPHYSICAL JOURNAL 73 1 357 - 366 1997年07月 [査読有り][通常論文]
   

  The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol, In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay, This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension, In the presence of ethanol, however, we observed the wavelength-dependent anisotropies, The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates, The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin, The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.
• Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane

  Takashi Kikukawa, Tsunchisa Araiso, Kôichi Mukasa, Tateo Shimozawa, Naoki Kamo

  Chemical and Pharmaceutical Bulletin 44 3 473 - 476 1996年 [査読有り][通常論文]
   

  We measured the absorption anisotropies of bacteriorhodopsin (bR) within a purple membrane suspension after photo-excitation in the millisecond time range. The purple membranes used were isolated from Halobacterium salinarium grown at three different culture temperatures, 37.0, 43.0 and 47.5 °C. For the membranes from the 37.0 °C culture, the observed anisotropies at wavelengths of 410, 570 and 680 nm showed almost the same slow decay. The slow decaying of the anisotropies originated from the rotation of the membrane itself. Using the membranes from the 43.0 and 47.5°C culture, however, we found that the anisotropy change varied at each wavelength measured. In these cases, it is shown from detailed data analysis that 1) the rotational motion of photo-intermediates within the membrane is more restricted than that of non-excited bR and 2) the distorted arrangements of the proteins within the membrane remain, even after photo-intermediates return to ground-state bR. This restricted motion is probably caused by the conformational changes in photo-intermediates, which prevent the rotation of the monomer protein and/or lead photo-intermediates to bind with neighboring proteins.
• The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane

  T Kikukawa, T Araiso, K Mukasa, T Shimozawa, N Kamo

  FEBS LETTERS 377 3 502 - 504 1995年12月 [査読有り][通常論文]
   

  We measured the flash-induced absorption anisotropies of mutant bacteriorhodopsin (bR), D96N, in the purple membrane suspension, The measured anisotropy decay at 410 nm differed from that at 570 Mn. These wavelength-dependent anisotropies show that the motion of absorption dipole of non-excited bR is faster than that of M-intermediate, The motion of non-excited bR is considered as the rotational motion of whole protein in the purple membrane, This fact suggests that the photo-excitation induces the conformational change of the protein and/or the inter-protein interaction within the membrane, which prevents the motion of M-intermediate.

書籍

• 創薬研究のための相互作用解析パーフェクト : 低中分子・抗体創薬におけるスクリーニング戦略と実例、in silico解析、一歩進んだ分析技術まで

  菊川峰志, 尾瀬農之, 金城政孝 (担当:分担執筆範囲:I-7 蛍光偏光測定)
  羊土社 2021年12月 (ISBN: 9784758122566) 365p
• Epigenetics to Optogenetics - A New Paradigm in the Study of Biology

  Tamogami, J., Kikukawa, T. (担当:分担執筆範囲:Functional Mechanism of Proton Pump-Type Rhodopsins Found in Various Microorganisms as a Potential Effective Tool in Optogenetics)
  IntechOpen 2021年06月
• Optogenetics: Light-Sensing Proteins and Their Applications in Neuroscience and Beyond

  Kikukawa, T (担当:分担執筆範囲:Chapter 4, Functional Mechanism of Cl−-Pump Rhodopsin and Its Conversion into H+ Pump)
  Springer 2021年01月
• 光と生命の事典

  菊川峰志 (担当:分担執筆範囲:プロテオロドプシン)
  朝倉書店 2016年03月 (ISBN: 4254171617) 422

  
• Optogenetics: Light-Sensing Proteins and Their Applications

  Kikukawa, T, Kamo, N, Demura, M (担当:分担執筆範囲:Chapter 4, Photochemistry of Halorhodopsin)
  Springer 2015年06月 (ISBN: 4431555153) 409

  
• 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, 菊川峰志, 加茂直樹 (担当:分担執筆範囲:ハロロドプシンの分子構造と機能解析)
  エヌ・ティー・エス 2013年 (ISBN: 9784864690713)
• Molecular Photochemistry - Various Aspects

  Kikukawa, T, Tamogami, J, Shimono, K, Demura, M, Nara, T, Kamo, N (担当:分担執筆範囲:Chapter 5, Photo-induced Proton Transfers of Microbial Rhodopsins)
  InTech 2012年

講演・口頭発表等

• Retinal conformations in microbial rhodopsins probed by Raman optical activity  [招待講演]

  Masashi Unno, Tomotsumi Fujisawa, Takashi Kikukawa

  19th International Conference on Retinal Proteins 2022年11月 口頭発表（招待・特別）
• A possible gate-opening mechanism of Indibacter alkaliphilus sodium pump rhodopsin  [招待講演]

  Kikukawa, T.

  19th International Conference on Retinal Proteins 2022年10月 口頭発表（招待・特別）
• フラッシュフォトリシス法を用いた微生物ロドプシンの光反応解析  [招待講演]

  菊川 峰志

  日本生物物理学会第58回年会 2020年09月 シンポジウム・ワークショップパネル（公募）
• 微生物ロドプシンのホモ多量体形成の機能的意義  [招待講演]

  菊川 峰志

  東京大学物性研究所ワークショップ「レチナールタンパク質の光機能発現の物理と化学」 2019年09月 口頭発表（招待・特別）
• Cyanobacterial rhodopsin having TSD motif  [招待講演]

  Kikukawa, T

  8th Asia and Oceania Conference on Photobiology 2017年11月 口頭発表（招待・特別） Seoul, Korea
• Light-induced conformational change of inward Cl- pump halorhodopsin  [招待講演]

  Kikukawa, T, Kamo, N, Demura, M

  13th International Conference on Flow Dynamics 2016年10月 口頭発表（招待・特別）
• 時間分解吸収分光で膜蛋白質の反応中間体を捕らえる  [招待講演]

  菊川 峰志

  第10回日本蛋白質科学会年会 2010年06月 シンポジウム・ワークショップパネル（指名）
• Important amino acid residues for function of halorhodopsin from Natronomonas pharaonis, a light-driven Cl- pump  [招待講演]

  Kikukawa, T, Kubo, M, Miyauchi, S, Kamo, N, Demura, M

  4th Asia Oceania Conference on Photobiology 2008年11月 シンポジウム・ワークショップパネル（指名） Varanasi, India
• On the sensory rhodopsins in halobacteria, extremely halophilic archaea  [招待講演]

  Kikukawa, T, Abe, T, Nakao, Y, Kamo, N

  15th Anniversary meeting for Korean society of photoscience 2008年09月 シンポジウム・ワークショップパネル（指名） Jeju, Korea
• Analysis of photo-induced proton uptake/release by bacteriorhodopsin and proteorhodopsin using a SnO2 transparent electrode  [招待講演]

  Tamogami, J, Kikukawa, T, Miyauchi, S, Kamo, N

  13th International Conference on Retinal Proteins 2008年06月 シンポジウム・ワークショップパネル（指名） Barcelona, Spain
• Importance of specific hydrogen bonds of archaeal rhodopsins for the binding with transducer protein: what happens when the transducer protein binds to an engineered pharaonis halorhodopsin  [招待講演]

  Kamo, N, Sudo, Y, Hasegawa, C, Miyauchi, S, Kikukawa, T

  12th International Conference on Retinal Proteins 2006年06月 シンポジウム・ワークショップパネル（指名） Awaji, Japan

その他活動・業績

• カルモジュリン融合タンパク質システムを使用した安定同位体標識セクロピンP1の大量発現とNMR構造解析

  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
• Functional significance of interhelical interactions between aspartic acid and cysteine residues in microbial rhodopsin

  Jun Tamogami, Takashi Kikukawa, Naoki Kamo Atlas of Science (Web) 2019年06月 [査読無し][通常論文]
  
• 部分フッ素化リン脂質F4-DMPC膜に再構成した膜タンパク質バクテリオロドプシンの2次元格子の安定性

  高橋浩, 吉野賢, 森田康平, 高木俊之, 横山泰範, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史, 園山正史, 園山正史 KEK Progress Report (Web) (2019-7) 2019年
• 擬環状エーテル型脂質のフッ素化が膜物性および膜タンパク質の構造・機能に及ぼす影響

  川原るい, 高木俊之, 高橋浩, 菊川峰志, 網井秀樹, 園山正史, 園山正史, 園山正史 フッ素化学討論会講演要旨集 42nd 2019年
• マウス抗菌ペプチドcryptdin‐4の変異体を用いた作用機構の解析

  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]
  
• ジャガイモ由来抗菌ペプチドsnakin‐1の発現系構築

  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]
  
• シマグワワインのNMRメタボローム解析

  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]
  
• ナトリウムポンプ型ロドプシンにおけるイオン結合サイトの役割

  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]
  
• ジャガイモ由来抗菌ペプチドsnakin‐1の安定的な発現・精製系の構築とNMRによる構造解析

  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]
  
• 部分フッ素化リン脂質膜に再構成したバクテリオロドプシンが形成する六方格子構造の熱安定性

  高橋浩, 吉野賢, 高木俊之, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史 日本物理学会講演概要集(CD-ROM) 71 (1) 2016年
• 部分フッ素化リン脂質の膜構造の特徴とその膜に再構成したバクテリオロドプシンの二次元格子の熱安定性

  高橋浩, 吉野賢, 高木俊之, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史 KEK Proceedings (Web) (2016-2) 2016年
• 新規部分フッ素化リン脂質ライブラリーの構築:膜タンパク質バクテリオロドプシン研究への応用

  吉野賢, 橋本麻美, 堀越未希, 村井裕佳, 森田康平, 柳瑶美, 茂木俊憲, 菊川峰志, 高木俊之, 高橋浩, 網井秀樹, 金森敏幸, 園山正史 フッ素化学討論会講演要旨集 39th 2016年
• 固体NMRによるタンパク質測定への圧縮センシングの応用

  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]
  
• 抗菌ペプチドthanatinとリポ多糖の相互作用解析

  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]
  
• 固体NMR法を用いたナトリウムポンプ製ロドプシンの構造解析

  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]
  
• 部分フッ素化リン脂質を利用した膜タンパク質研究の展開

  園山正史, 吉野賢, 村井裕佳, 森田康平, 菊川峰志, 高木俊之, 高橋浩, 網井秀樹, 網井秀樹, 金森敏幸 日本蛋白質科学会年会プログラム・要旨集 15th 2015年
• 疎水鎖末端をパーフルオロアルキル基に置換したDMPC脂質二分子膜中の膜タンパク質の構造・機能・安定性 2.DMPCを含む二成分系混合膜におけるバクテリオロドプシンの分配挙動

  堀越未希, 吉野賢, 金山賢治, 森田康平, 高橋浩, 網井秀樹, 菊川峰志, 高木俊之, 金森敏幸, 園山正史 分子科学討論会講演プログラム&要旨(Web) 9th 2015年
• 疎水鎖末端をパーフルオロアルキル基に置換したDMPC脂質二分子膜中の膜タンパク質の構造・機能・安定性 1.パーフルオロアルキル基によるバクテリオロドプシンの安定化

  橋本麻美, 村井裕佳, 森田康平, 高橋浩, 網井秀樹, 菊川峰志, 高木俊之, 金森敏幸, 園山正史 分子科学討論会講演プログラム&要旨(Web) 9th 2015年
• 高圧閃光光分解法を用いた光駆動性アニオンポンプ:ハロロドプシンのCl^-輸送機構の解析

  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]
  
• 膜タンパク質ASRとの相互作用解析に向けたASRTのNMR解析

  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]
  
• 常磁性緩和促進を利用したDNA結合ドメインSTPRの分子ダイナミクスの解析

  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]
  
• 固体NMRにおける常磁性緩和促進を用いたタンパク質立体構造解析

  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]
  
• 再構成無細胞系を用いた短鎖ペプチドの直接発現

  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]
  
• 二量体化したDi-O-Tetradecylphosphatidylcholineへの膜タンパク質バクテリオロドプシンの再構成

  土田直之, 高木俊之, 菊川峰志, 高橋浩, 金森敏幸, 園山正史 日本化学会講演予稿集 94th (3) 2014年
• 固体NMRにおけるGFT NMRを応用したタンパク質連鎖帰属法ならびに解析支援プログラムの開発

  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]
  
• 固体NMRによるタンパク質の構造解析に向けた¹³C‐常磁性緩和促進の研究

  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]
  
• 部分フッ素化リン脂質を含む二成分系リン脂質混合膜における膜タンパク質バクテリオロドプシンの分配挙動

  吉野賢, 金山賢治, 高橋浩, 高木俊之, 菊川峰志, 横山泰範, 網井秀樹, 金森敏之, 園山正史 生体分子科学討論会講演要旨集 40th 84 -85 2013年06月07日 [査読無し][通常論文]
  
• CD and NMR Analysis of a Peptide Mimicking the First Extracellular Loop of the Transmembrane Protein Halorhodopsin

  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
• Importance of Salt Bridge in Cryptdin-4 for Folding in Denatured Condition

  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
• NMR Structural Analysis of Potato Antimicrobial Peptide Snakin-1

  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
• 部分フッ素化リン脂質・非フッ素化リン脂質二成分混合膜に再構成したバクテリオロドプシンの脂質選択性

  吉野賢, 菊川峰志, 高橋浩, 高橋浩, 高木俊之, 網井秀樹, 網井秀樹, 金森敏幸, 園山正史, 園山正史 日本蛋白質科学会年会プログラム・要旨集 13th 2013年
• 部分フッ素化リン脂質膜中に再構成したバクテリオロドプシンの光サイクルと二次元格子構造について

  高橋浩, 吉野賢, 菊川峰志, 高木俊之, 横山泰範, 網井秀樹, 馬場照彦, 金森敏幸, 園山正史 物構研サイエンスフェスタ要旨集 1st 57 2013年 [査読無し][通常論文]
  
• 牛α‐スペクトリンの遺伝子多型(E91K置換)による分子構造と赤血球膜物性の異常

  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]
  
• カブトガニ由来抗菌ペプチド Tachyplesin I のキチン結合能

  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]
  
• 新規部分フッ素化リン脂質に再構成したバクテリオロドプシンに対する脂質フッ素化の影響

  吉野賢, 高木俊之, 菊川峰志, 高橋浩, 馬場照彦, 金森敏之, 園山正史 生体分子科学討論会講演要旨集 39th 38 -39 2012年06月08日 [査読無し][通常論文]
  
• ハロロドプシンの光反応サイクル中に起こるカロテノイドの吸光度変化

  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]
  
• 牛α‐スペクトリン・アレルSpαBK91に起因するリピート1の構造異常と赤血球膜物性の変化

  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]
  
• NMR Structure and Interaction of Antimicrobial Peptide Tachyplesin I with Lipopolysaccharide

  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
• Does Escherichia coli Thioredoxin Improve Expression Efficiency of Recombinant Peptide in Pichia pastoris?

  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
• Structural Analysis of Plant Antimicrobial Peptide Snakin-1

  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
• Expression and Structural Analysis of a Recombinant Antibacterial Peptide, Cecropin P1

  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
• Expression, Purification and NMR Characterization of the Cyclic Reconbinant Form the First Extracellular Loop of the Transmembrane Protein Halorhodopsin

  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
• 三次元固体NMR法による膜タンパク質ハロロドプシンの研究

  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
• 固体NMRにおけるタンパク質連鎖帰属へのGFT NMR法の応用

  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
• Conformational analysis of novel peptide derivatives with cytostatic activity

  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
• 非線形サンプリングを用いた膜タンパク質の固体NMR測定

  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
• 新規微生物型ロドプシン(MR)から観る光駆動イオンポンプから光センサーへの分子進化

  須藤雄気, 井原邦夫, 小林詩織, 鈴木大介, 入枝泰樹, 菊川峰志, 神取秀樹, 本間道夫 生化学 ROMBUNNO.2T16A-2 2011年 [査読無し][通常論文]
  
• Exploring the active site structure of photoreceptor proteins by Raman optical activity

  Masashi Unno, Takahito Shingae, Takashi Kikukawa, Naoki Kamo AIP Conference Proceedings 1267 102 -103 2010年12月14日 [査読無し][通常論文]
  
• 固体NMR法による7TMハロロドプシン膜外ループの構造解析

  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
• 膜タンパク質ハロロドプシンの多次元固体NMR-同位体ラベルの最適化-

  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
• 2P302 アザイド存在下におけるハロロドプシンのプロトン輸送機構の解析(生体膜・人工膜(輸送),ポスター発表,第45回日本生物物理学会年会)

  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]
  
• 3P223 浸透圧を加えた条件下におけるハロロドプシンの光化学反応(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

  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]
  
• 3P222 光駆動アニオンポンプ・ハロロドプシンのArg123の役割(光生物学(視覚と光受容)、放射線生物学,口頭発表,第45回日本生物物理学会年会)

  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]
  
• 1P268 ファラオニスハロロドプシンのクロライド放出側のArgの役割(光生物 A) 視覚・光受容))

  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]
  
• 1P271 ハロロドプシンCP側チャネル変異体K215Rの光中間体への寄与(光生物 A) 視覚・光受容))

  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]
  
• Roles of Ser130 and Thr126 in Chloride Binding and Photocycle of pharaonis Halorhodopsin

  Sato Maki, Kikukawa Takashi, Araiso Tsunehisa, OKITA Hirotaka, SHIMONO Kazumi, KAMO Naoki, DEMURA Makoto, NITTA Katsutoshi The journal of biochemistry 134 (1) 151 -158 2003年 [査読無し][通常論文]
  

受賞

• 2004年06月 産学連携学会 学会賞
  

共同研究・競争的資金等の研究課題

• 過渡現象解析で追求する膜輸送タンパク質の多段階構造変化と輸送素過程の連関

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 菊川 峰志
• 光エネルギーで駆動される物質回収・放出カプセルのボトムアップ構築

  日本学術振興会：科学研究費助成事業 学術変革領域研究(A)

  研究期間 : 2022年06月 -2024年03月 

  代表者 : 菊川 峰志
• アルブミン介在肝取り込み促進機構の解明とその促進機構モデルの新展開

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 宮内 正二, 菊川 峰志

   

  基礎学問から応用学問まで薬学領域において幅広く受け入れられている概念”free drug theory (FDT)”に従わない、アルブミン介在取り込み促進機構解明を詳細に解析するための最適な実験系の構築を行った。先ず、アフリカツメガエル卵母細胞発現系を用いて、アルブミン結合率の高い薬物を輸送する有機アニオン輸送担体（OATP,OAT）の輸送活性を示す発現系を確立した。構築した輸送担体発現系は高い輸送活性を示し、アルブミン存在下でも詳細な速度論的な解析が可能なものである。これら発現系はイオン駆動力が解明されていない有機アニオン輸送担体の分子輸送機構の解明、エネルギー共役系の解明にも応用できる有用な実験系であることが明らかになった。実際に、アルブミン存在下で輸送促進機構、特に、アルブミンの輸送駆動力に及ぼす影響について検討を進めている。更に、これまでアルブミン促進機構が報告されている臓器の抽出RNAを用いて有機アニオン輸送担体の単離、輸送実験系を確立した。これにより脳毛細血管や腎毛細血管において報告されているアルブミン促進機構の詳細なメカニズムとその生理的意義を今後検討する。 一方、並行して進めている蛍光物質利用した蛍光相関分光分析法（Fluorescence Correlation Spectroscopy：FCS）によりアルブミン介在肝取り込み促進を詳細に解析出来る実験系の構築を行っている。今年度明らかにした知見として、蛍光強度の自己相関関数の測定には、蛍光物質の励起状態安定性が必須であること、アルブミン促進を示す蛍光物質はFCSには適切で無いことである。今後、FCS解析に適切な蛍光物質の検討を現在行っている。
• 膜輸送タンパク質の光エネルギー共役を基盤とする環境浄化大腸菌の開発

  公益財団法人 住友財団：環境研究助成

  研究期間 : 2021年11月 -2022年11月 

  代表者 : 菊川 峰志
• 毒物耐性スクリーニングを用いた膜輸送タンパク質の革新的機能改変

  公益財団法人 秋山記念生命科学振興財団：一般助成

  研究期間 : 2021年04月 -2022年03月 

  代表者 : 菊川 峰志
• 多彩な基質輸送能力を有する輸送担体を制御する輸送分子機構解明

  文部科学省：科学研究費補助金（基盤研究(C)）

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 宮内 正二
• 光エネルギーによる環境浄化微生物の設計

  日本学術振興会：科学研究費助成事業

  研究期間 : 2016年04月 -2019年03月 

  代表者 : 出村 誠, 菊川 峰志

   

  環境を光によって浄化する大腸菌の作製を目指して、光駆動型イオンポンプ膜蛋白質である微生物型ロドプシンと、様々な薬物を膜輸送できる多剤排出蛋白質を大腸菌細胞内で共役させ、薬物輸送を光によって駆動することを試みた。その結果、大腸菌内では、Na+を介した共役が有利であることを見出した。また、環境浄化大腸菌作製に有用と考えられる、強い内向きH+ポンプ活性を持った微生物型ロドプシンを南極で単離された真正細菌から見出した。
• 光駆動型ナトリウムポンプ：イオン輸送素過程の解明と機能改変の試み

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2017年 -2019年 

  代表者 : 菊川 峰志
• 光機能性バイオナノマテリアルの2光子吸収過程―機構解明とデバイス開発に向けて―

  日本学術振興会：科学研究費助成事業

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 横山 泰範, 岸田 英夫, 園山 正史, 高橋 浩, 菊川 峰志, 竹中 康司, 田中 輝, 茂木 健太郎, 矢野 俊介

   

  光機能性のバイオマテリアル・バクテリオロドプシン(bR)を用いた新規な光書込みデバイス開発にとって、核となる技術であるbRの固体化に取組んだ。bRを光記録デバイスに応用するためには、固体中でもbRの光反応による光書込みが可能で、かつ高密度書込みのために記録層(bR膜)の積層が重要となる。親水性高分子であるポリビニルアルコール(PVA)を固定媒体として作製した固体試料に対し、bRの機能・構造上の特徴を検討した。固体試料中のbRは溶液系とほぼ同一の光応答を示し、かつ天然で形成する高次構造を維持していた。また、PVAのゲル化に伴いbR膜の積層が起き、PVA系は将来的な応用に有用であることがわかった。
• トランスポーターの基質輸送サイクルを決定する分子弁の同定と機能解析

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2014年 -2016年 

  代表者 : 宮内 正二

   

  本研究では、トランスポーターの基質輸送サイクルを決定する分子弁の同定とその輸送分子機構を明らかにすることを目的として、様々な起電性輸送担体をモデル輸送担体として用い、以下に示す研究成果を得た。 (1) ヒトH+/オリゴペプチド共輸送担体 (hPEPT1)の基質輸送の方向性を決定する分子弁の実体を明らかにした。 (2) Na+/モノカルボン酸共輸送担体 (hSMCT1) の方向性を決定する分子弁の存在を明らかにした。
• 光駆動型ナトリウムポンプの生理的意義とイオン輸送機構の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2014年 -2016年 

  代表者 : 菊川 峰志

   

  微生物型ロドプシンは、色素レチナールを内包する膜タンパク質である。レチナールの光吸収をきっかけとして、複数の構造の異なる中間体を経由する光反応サイクルを周り、この間にイオンの膜輸送や光情報伝達を行う。本研究では、外向きにNa+を輸送するNa+ポンプ型ロドプシン（NaR）の機能解析を行い、以下の成果を得た。１）タンパク質表面へのカチオン結合が暗状態の構造を変調し、光反応初期に出現する中間体を決定する。２）NaRのNa+取込みと放出が、O中間体の生成・崩壊に同期して起こることを、Na+濃度変化の直接観測から明らかとした。
• イオン輸送を伴うトランスポーターの輸送分子機構の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2011年 -2013年 

  代表者 : 宮内 正二, 菊川 峰志

   

  H+ 濃度勾配を利用してジペプチドから医薬品まで能動輸送するH+/オリゴペプチド輸送担体 (PEPT) の輸送分子機構の解明をおこなった．PEPT は，第二膜貫通領域 (TDM2) に存在する輸送活性中心である His57 のプロトン化により活性調節されているが，どの様なメカニズムで活性調節をおこなっているか不明である．His57のプロトン化による輸送活性の制御がどの様な分子メカニズムによるかを検討するために，PEPTsoの結晶構造（バクテリアにおけるPEPT類似輸送担体）を基にHis57と相互作用する可能性が高いSer302の変異体を作成し，輸送活性のpH依存性を検討した．結晶構造情報によれば，His57とSer302との距離は水素結合が可能な2.7Åにある． Ser302をAla, Thr, Valに変化させた変異体をアフリカツメガエル卵母細胞に発現させGly-Sar (典型的なPEPTの基質)の取り込み輸送活性のpH依存性を検討した． Ala, Thrの変異体は野生型hPEPT1に比べて輸送活性は低下したが，輸送活性は保持していた．取り込み輸送活性のpH依存性は，Ala, Thrの変異体について，ベル型の至適pHがアルカリ側にシフトした．S302V変異体についてはHis57が水素結合を形成することが出来ない状態にあり，輸送活性が見られなかった．様々な代謝酵素において，活性中心のSerの水酸基がHisのイミダゾ-ル基と水素結合を形成し，His残基側鎖のプロトン化に伴う水素結合ネットワーク変化により，活性調節がなされている．Gly-Sar輸送活性のpH依存性のアルカリ側へのシフトは，hPEPT1にもよく似たpH活性調節機構が備わっておりHis57とSer302の水素結合が活性調節に重要であるという可能性を示唆したものである．
• 光駆動型イオンポンプ：ハロロドプシンのクロライド輸送素過程の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2011年 -2013年 

  代表者 : 菊川 峰志, 宮内 正二

   

  高度好塩菌の細胞膜に存在するハロロドプシン（HR)は、光エネルギーを用いて、Cl-を細胞外から細胞内へ輸送する蛋白質である。光で活性化できるという利点に加え、輸送活性などの測定手法の確立によって、HRは輸送分子機構を詳細に解析できる優れたモデル蛋白質となってきている。本研究では、輸送に伴う分子内イベントの解析と、そこに係わる重要アミノ酸残基の同定を通して、HRの分子機構を考察する。今年度は、以下の二つの成果を得た。１．HRが光反応サイクル中に発生する起電力の時間分解測定：誘電性薄膜であるコロジオン膜にHRを吸着させ、この膜を用いた電気化学セルを構築した。誘電性薄膜はコンデンサ、HRは電池とみなせるため、Cl-輸送に伴う輸送起電力は、光誘導電流として観測される。電気化学セルにナノ秒パルスレーザを照射したところ、数ミリ秒で最大となる誘導電流を観測した。これは、光反応サイクルにおけるO中間体の生成・崩壊のタイミングとほぼ一致しており、この中間体の前後で、Cl-の長距離の移動が起こることが示唆された。２．H.salinarum由来のHR（HsHR）の大腸菌発現系を用いた調製法の確立：HRの研究には、HsHRとN.pharaonis由来のHR（NpHR）が主に用いられてきた。両者には、輸送基質の種類や光反応サイクルなど、興味深い違いがあることが知られており、これらを比較することで、Cl-輸送機構をより詳細に検討できるものと期待される。NpHRは、大腸菌における機能的大量発現系が確立しているが、HsHRでは、これがなかった。そこで、HsHRの大腸菌発現系を用いた調製を試みた。その結果、HsHRは、不活性な状態で大腸菌膜に発現するが、可溶化時にレチナールを添加することで、活性型のHsHRを得られることが明らかとなった。この手法によって、HsHR変異体を容易に調製できるようになった。
• トランスポーターの輸送分子機構解明の新展開:光駆動性ポンプをモデルタンパクとして研究

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2008年 -2010年 

  代表者 : 宮内 正二, 菊川 峰志

   

  光駆動性クロライドポンプの輸送分子機構を明らかにすることを研究目的として研究を行い,輸送活性を様々な物理化学的手法より測定し,以下の結果を得た.(1)種々の変異体を作成し,その変異体におけるCl-結合能を測定した.結合に対して重要な残基を特定した.Arg123,Ser130,Asp252である.(2)Cl^-の細胞外から細胞内へのタンパク内移動には,Ser130およびAsp252が重要であることが明らかとなった.(3)Ser130は,Cl^-が細胞質側から細胞外側に逆流が起こらないようにする分子弁として機能していることが明らかとなった
• 光駆動型アニオンポンプ蛋白質:ハロロドプシンの分子機構

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2007年 -2009年 

  代表者 : 菊川 峰志, 宮内 正二

   

  高度好塩菌の細胞膜に存在するハロロドプシンは、光エネルギーを用いて、Cl-イオンを細胞外から細胞内へ輸送するイオンポンプ膜タンパク質である。その分子機構解明に寄与するため、野生型、及び、アミノ酸置換した複数の変異体を用いた解析を行い、以下の知見を得た。(1)活性中心近傍の酸性及び芳香族アミノ酸残基は、輸送の初期過程において重要な役割を果たす。(2)輸送サイクル中に現れる、Cl-濃度依存的な二つの中間体の平衡状態は、Cl-放出過程を反映している。(3)Cl-放出時には、蛋白質の細胞質側チャネルが水和し、Cl-放出状態が安定化する。
• ハロロドプシンのクロライド輸送機構解明と3量体形成の意義

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2007年 -2009年 

  代表者 : 加茂 直樹, 宮内 正二, 鷲見 正人, 菊川 峰志, 奈良 敏文, 宮内 正二

   

  ハロロドプシン(HR)は高度好塩菌の膜に発現している光エネルギーで駆動されるClイオンポンプである。Xenopusオーサイトに発現させ、光照射による膜電流の測定より、活性を測定する新しい方法を開発した。非常に定量性優れていた。大腸菌の発現系を用い、種々のHRの変異体を作成し、活性と光化学反応を調べた。Arg123が輸送に重要であることを明らかにした。NからO中間体への変化の際に、ClイオンがHR内部から細胞内部へ放出され、O→HR'の過程でClが細胞外からHRへ取り込まれることを明らかにした。HRは界面活性剤中でも3量体を形成していることを明らかにした。3量体はモノマーより安定であった。
• アフリカツメガエル卵母細胞を用いた電位発生的輸送担体研究の新しい展開

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2006年 -2007年 

  代表者 : 宮内 正二, 菊川 峰志

   

  1.光駆動性クロライドポンプ(HR)のクロライド輸送機構の解明光駆動性クロライドポンプ(HR)のクロライド輸送機構を電気生理学的に精密に検討することが出来る系を確立した.世界ではじめてこの実験に成功している.この実験の他では出来ないユニークな点は,膜電位をかけながら,Cl^-の輸送が実験出来ることである.これを利用して,HR1分子当たりの「輸送能力」と1秒間に輸送するイオン輸送能力を見積もることを可能とした.これで,様々な変異体の輸送活性を精度良く見積もることができるようになった.輸送における輸送中間体の役割を明らかにするために最も重要な事柄のひとつである.2.オリゴペプチド輸送担体(hPEPT1)の基質輸送機構の解明hPEPT1がトランスポーターでありながら,特殊な基質存在下では,基質認識部位における構造変化によりH^+チャンネルとしても働き得ること見いだした.この知見により、トランスポーターは(チャネル)+(基質スイッチ)の2つの機構からなり,これらを協調的に機能させ,基質を輸送するという作業仮説に至った.この性質は,上紀のCl_-の膜透過過程にも含まれ,膜透過の本質であることも明らかになった.3.ヒトNa^+/モノカルボン酸共輸送体(hSMCT)の基質認識機構の解明構造の異なるモノカルボン酸を用いてhSMCTの斯質認識に関する法則を明らかにした.化合物が長いほど親和性は...
• 抗菌薬排出蛋白質の分子機構-細菌の小型排出蛋白質を例として-

  文部科学省：科学研究費補助金(若手研究(B))

  研究期間 : 2004年 -2006年 

  代表者 : 菊川 峰志

   

  ほぼ全ての生物種の細胞膜には,細胞にとって都合の悪い様々な毒物を認識し,能動的に細胞外へと排出する多剤排出蛋白質が存在している.これらの蛋白質は,感染症治療の大きな障害である多剤耐性菌株の出現と密接に係わっている他,分子科学の面からも,構造上相関の無い多様な薬物の認識/排出という興味深い機能を実現している.本研究では,多剤排出蛋白質の中で,最もサイズが小さいSMR(small multidrug resistance)ファミリーに属する蛋白質の分子機構解明を目指した研究を行ってきた.様々な測定手法を適用するためには,蛋白質の高純度な精製が必要なため,SMR蛋白質の大腸菌における大量発現・精製を重要な課題と位置付けて研究を行った.融合タグを用いる方法で,大腸菌のEmrEの大量発現と融合蛋白質の精製は実現できたが,残念ながら,融合タグの分離が依然として困難であった.有機溶媒に溶けるほどのSMRの脂溶性の高さが,プロテアーゼ処理とその後の蛋白質の回収を困難にしていると考えられる.一方,これと平行して,枯草菌のEbrABの膜内トポロジーの決定と,その制御機構を明らかとする研究を行い,興味深い知見を得るに至った.これまでのSMRの研究は,主にEmrEを用いて行われてきた.膜内ではホモダイマーとして機能すると考えられているが,その膜内トポロジーについては,互いに異なる報告がされてきている...
• 膜蛋白複合体における情報伝達機構:高度好塩菌の光受容を例として

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2004年 -2006年 

  代表者 : 加茂 直樹, 奈良 敏文, 宮内 正二, 菊川 峰志, 鷲見 正人

   

  生体膜を介する諸機能において,蛋白質複合体で機能する例は多くある.その1つの例として,本研究では高度好塩菌の走光性の受容体(フォボロドプシン,または,センソリーロドプシン)および受容体とトランスジューサ蛋白との相互作用の研究を行った.受容体とトランスジューサは共に膜蛋白である.得られた結果は次の通りである.1.微生物ロドプシンでは,シッフ塩基の近傍に存在するアルギニンは完全に保存されている.このアルギニンの変異体を作成し,この残基の役割を検討した.2.容体が光を受けると,Fヘリックスのコンフォメーションが変化することを示した.3.FTIRの結果から,受容体分子の中の水分子の水素結合が変化することを示した.4.固体NMRの手法を用いて,トランスジューサのダイナミクス変化が,受容体であるフォボロドプシンと結合すると変化することを示した.5.酸化スズの透明電極を用いて,フォボロドプシンの光照射に伴うプロトンの移動を調べ,Asp193からプロトンが放出される事およびこの残基のpKaがトランスジューサとの結合によって変化することを示した.6.トランスジューサの細胞質内の可溶性部分が受容体であるフォボロドプシンと相互作用していることをしました.7.両膜蛋白は膜内で3本の水素結合で相互作用していることを,変異体の結合実験から示した.また,両蛋白のvan deer Waals相互作用も結合に...
• Molecular Mechanism of Multidrug Transporter

  Grant-in-Aid for Scientific Research

  研究期間 : 2004年
• アラメシチンのチャネル形成過程-膜脂質の動的構造変化の解析から-

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1998年 -1999年 

  代表者 : 菊川 峰志

   

  アラメシチンは生体膜に取り付き、膜に電位差が加わると数分子が会合したチャネルを形成する抗生ペプチドである。チャネルの開状態モデル-Barrel Stave Model-は広く受け入られている。しかし、このペプチドがチャネル形成前(閉状態)に膜のどの部分(深さ方向)にどのような構造をとって取り付いているのか、また、どのようなメカニズムで膜を貫通するようになるのかという問題は未だ解決されていない。この問題の解明に、「アラメシチンと脂質との相互作用」という新しい視点に立ち、昨年度から、主に蛍光性脂質を導入したリポソームを用いて取り組んできた。今年度は、液晶相での脂質の頭部及び鎖部の運動性が、アラメシチンにより修飾される様子の詳細な検討を試みた。得られた知見をまとめると以下のようになる。1.脂質運動性の修飾はあるアラメシチン濃度を境に急峻になる。アラメシチンのcooperativityが反映していると考えられる。アラメシチンの侵入が脂質の相構造に大きな擾乱となっていることがわかる。2.脂質組成によって脂質の頭部及び鎖部の運動が異なる修飾を受ける。これは、アラメシチンの存在位置がアラメシチン濃度とともに変化していることを示している。3.脂質運動性の修飾は、微小量のアラメシチン濃度(脂質の3/100程度)で見られる。したがって、アラメシチンは近傍の脂質分子とだけ相互作用するわけではなく、...
• Molecular Mechanism of Microbial Rhodopsins

  Grant-in-Aid for Scientific Research

  研究期間 : 1999年

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• 2023年01月 - 現在   日本生物物理学会   生物物理編集委員
• 2020年04月 - 現在   日本生物物理学会   分野別専門委員
• 1998年08月 - 1999年03月   北海道立工業試験場   特許情報移転促進委員