MF研究者総覧

Researcher Database

Advance
Researchers' Page on researchmap
Last Updated :2024/11/01

Researcher Profile and Settings

Master

Affiliation (Master)

  • Faculty of Advanced Life Science　Functional Life Sciences　Biological Information Analysis Science

Affiliation (Master)

  • Faculty of Advanced Life Science　Functional Life Sciences　Biological Information Analysis Science

researchmap

Profile and Settings

Degree

  • Ph.D.（Hokkaido University）

Profile and Settings

  • Name (Japanese)

    Tsukamoto

  • Name (Kana)

    Takashi

  • Name

    201701018533416257

Alternate Names

Achievement

Research Interests

  • Ion pump   Ion channel   過渡吸収分光   膜輸送   Membrane Proteins   Rhodopsin   Photobiology   

Research Areas

  • Life sciences / Functional biochemistry / Ion channel, Ion pump
  • Life sciences / Ecology and environmental science / Extreme environmental microorganisms
  • Life sciences / Biophysics / Optogeneticd
  • Nanotechnology/Materials / Molecular biochemistry
  • Life sciences / Biophysics / Photobiology

Research Experience

  • 2017/04 - Today Hokkaido University Faculty of Advanced Life Science Assistant professor
  • 2014/07 - 2017/03 Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Assistant professor
  • 2014/04 - 2014/06 Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
  • 2013/04 - 2014/03 Nagoya University Division of Biological Science, Graduate School of Science
  • 2010/09 - 2010/11 National Institute for Materials Science

Education

  • 2010/04 - 2013/03  Hokkaido University  Graduate School of Life Science
  • 2008/04 - 2010/03  Hokkaido University  Graduate School of Life Science
  • 2004/04 - 2008/03  Hokkaido University  School of Science  Biological Sciences (Macromolecular Functions)

Committee Memberships

  • 2020/01 -2023/12   The Biophysical Society of Japan   Member, Specialized technical committee

Awards

  • 2005/10 Hokkaido University Nitobe Prize
     
    受賞者: TSUKAMOTO Takashi

Published Papers

  • Kaori Kondo, Ryouhei Ohtake, Shunsuke Nakano, Mia Terashima, Hisaya Kojima, Manabu Fukui, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
    Biochemistry 2024/09/17
  • Chihaya Hamada, Keisuke Murabe, Takashi Tsukamoto, Takashi Kikukawa
    The Journal of biological chemistry 300 (9) 107712 - 107712 2024/09 
    Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.
  • 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 2023/10/25 [Refereed]
     
    Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin (NaR), which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of NaR from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.
  • Yuya Ohki, Tsukasa Shinone, Sayo Inoko, Miu Sudo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
    The Journal of biological chemistry 105305 - 105305 2023/09/29 [Refereed]
     
    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.
  • Masaiku Ohya, Takashi Kikukawa, Junpei Matsuo, Takashi Tsukamoto, Ryota Nagaura, Tomotsumi Fujisawa, Masashi Unno
    The journal of physical chemistry. B 2023/05/18 [Refereed]
     
    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.
  • Kunisato Kuroi, Takashi Tsukamoto, Naoya Honda, Yuki Sudo, Yuji Furutani
    Biochimica et biophysica acta. Bioenergetics 1864 (3) 148980 - 148980 2023/04/18 [Refereed]
     
    The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to β-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to β-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.
  • Yuhei Doi, Jo Watanabe, Ryota Nii, Takashi Tsukamoto, Makoto Demura, Yuki Sudo, Takashi Kikukawa
    Scientific reports 12 (1) 16422 - 16422 2022/09/30 [Refereed]
     
    Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42-, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42- pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42- transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42- pump activity. Notably, the MrHR and SyHR mutants did not show SO42- induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42- in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42- into their centers after illumination and release it before the ends of the photoreactions.
  • Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa
    ACS Omega 2470-1343 2022/09/13 [Refereed]
  • Tomoya Kato, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
    The Journal of biological chemistry 100792 - 100792 2021/05/18 [Refereed]
     
    Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters due to the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+ pump rhodopsin (NaR). We triggered the transport cycle of NaR using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions, and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.
  • Chihiro Kikuchi, Hina Kurane, Takuma Watanabe, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
    Scientific reports 11 (1) 7908 - 7908 2021/04/12 [Refereed]
     
    Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO3- of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO3- transport and might take part in NO3--related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3rd transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO3-. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.
  • Keisuke Murabe, Takashi Tsukamoto, Tomoyasu Aizawa, Makoto Demura, Takashi Kikukawa
    Journal of the American Chemical Society 142 (37) 16023 - 16030 2020/09/06 [Refereed][Not invited]
     
    For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle.
  • Ayumi Yamamoto, Takashi Tsukamoto, Kenshiro Suzuki, Eri Hashimoto, Yoshihiro Kobashigawa, Kousuke Shibasaki, Takeshi Uchida, Fuyuhiko Inagaki, Makoto Demura, Koichiro Ishimori
    Biophysical journal 118 (11) 2853 - 2865 2020/04/29 [Refereed][Not invited]
     
    We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.
  • Satoshi Yasuda, Tomoki Akiyama, Sayaka Nemoto, Tomohiko Hayashi, Tetsuya Ueta, Keiichi Kojima, Takashi Tsukamoto, Satoru Nagatoishi, Kouhei Tsumoto, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata
    Journal of chemical information and modeling 60 (3) 1709 - 1716 2020/03/23 [Refereed][Not invited]
     
    We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.
  • Azusa Iizuka, Kousuke Kajimoto, Tomotsumi Fujisawa, Takashi Tsukamoto, Tomoyasu Aizawa, Naoki Kamo, Kwang-Hwan Jung, Masashi Unno, Makoto Demura, Takashi Kikukawa
    Scientific Reports Springer Science and Business Media {LLC} 9 (1) 2045-2322 2019/12 [Refereed][Not invited]
     
    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.
  • Taro Yamanashi, Misayo Maki, Keiichi Kojima, Atsushi Shibukawa, Takashi Tsukamoto, Srikanta Chowdhury, Akihiro Yamanaka, Shin Takagi, Yuki Sudo
    Scientific Reports 9 (1) 2045-2322 2019/03 [Refereed][Not invited]
  • Takashi Tsukamoto, Chihiro Kikuchi, Hiromu Suzuki, Tomoyasu Aizawa, Takashi Kikukawa, Makoto Demura
    Scientific Reports 8 (1) 13445 - 13445 2018/12 
    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-.
  • Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani
    The Journal of Physical Chemistry B American Chemical Society ({ACS}) 122 (42) 9826 - 9826 2018/10/25 [Refereed][Not invited]
  • Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani
    The Journal of Physical Chemistry B American Chemical Society ({ACS}) 122 (27) 6945 - 6953 1520-6106 2018/07 [Refereed][Not invited]
  • Riho Takayama, Akimasa Kaneko, Takashi Okitsu, Satoshi P. Tsunoda, Kazumi Shimono, Misao Mizuno, Keiichi Kojima, Takashi Tsukamoto, Hideki Kandori, Yasuhisa Mizutani, Akimori Wada, Yuki Sudo
    Journal of Physical Chemistry Letters 9 (11) 2857 - 2862 1948-7185 2018/06/07 [Refereed][Not invited]
     
    Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.
  • 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 2018/03/29 [Refereed][Not invited]
     
    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.
  • Saki Inoue, Susumu Yoshizawa, Yu Nakajima, Keiichi Kojima, Takashi Tsukamoto, Takashi Kikukawa, Yuki Sudo
    Physical chemistry chemical physics : PCCP 20 (5) 3172 - 3183 2018/01/31 [Refereed][Not invited]
     
    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.
  • Naoya Honda, Takashi Tsukamoto, Yuki Sudo
    CHEMICAL PHYSICS LETTERS 682 6 - 14 0009-2614 2017/08 [Refereed][Not invited]
     
    Rhodopsins are seven-transmembrane proteins that function as photoreceptors for a variety of biological processes. Their characteristic visible colors make rhodopsins a good model for membrane-embedded proteins. In this study, by utilizing their color changes, we performed comparative studies on the stability of five microbial rhodopsins using the same instruments, procedures and media. As denaturants, we employed four physicochemical stimuli: (i) thermal perturbation, (ii) the water-soluble reagent hydroxylamine, (iii) the detergent sodium dodecyl sulfate, and (iv) the organic solvent ethanol. On the basis of the results, models for stabilization mechanisms in rhodopsins against each stimulus is proposed. (C) 2017 Elsevier B.V. All rights reserved.
  • Takashi Tsukamoto, Susumu Yoshizawa, Takashi Kikukawa, Makoto Demura, Yuki Sudo
    JOURNAL OF PHYSICAL CHEMISTRY B 121 (9) 2027 - 2038 1520-6106 2017/03 [Refereed][Not invited]
     
    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.
  • Akiko Niho, Susumu Yoshizawa, Takashi Tsukamoto, Marie Kurihara, Shinya Tahara, Yu Nakajima, Misao Mizuno, Hikaru Kuramochi, Tahei Tahara, Yasuhisa Mizutani, Yuki Sudo
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 139 (12) 4376 - 4389 0002-7863 2017/03 [Refereed][Not invited]
     
    In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+ and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 rriM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics.
  • Kanae Kanehara, Susumu Yoshizawa, Takashi Tsukamoto, Yuki Sudo
    SCIENTIFIC REPORTS 7 44427  2045-2322 2017/03 [Refereed][Not invited]
     
    Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941(T) that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pK(a) values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times).
  • Satoko Doi, Takashi Tsukamoto, Susumu Yoshizawa, Yuki Sudo
    SCIENTIFIC REPORTS 7 41879  2045-2322 2017/02 [Refereed][Not invited]
     
    Anion channelrhodopsin-2 (ACR2) was recently identified from the cryptophyte algae Guillardia theta and has become a focus of interest in part because of its novel light-gated anion channel activity and its extremely high neural silencing activity. In this study, we tried to express ACR2 in Escherichia coli cells as a recombinant protein. The E. coli cells expressing ACR2 showed an increase in pH upon blue-light illumination in the presence of monovalent anions and the protonophore carbonyl cyanide mchlorophenylhydrazone (CCCP), indicating an inward anion channel activity. Then, taking advantage of the E. coli expression system, we performed alanine-scanning mutagenesis on conserved basic amino acid residues. One of them, R84A, showed strong signals compared with the wild-type, indicating an inhibitory role of R84 on Cl-transportation. The signal was strongly enhanced in R84E, whereas R84K was less effective than the wild-type (i.e., R84). These results suggest that the positive charge at position 84 is critical for the inhibition. Thus we succeeded in functional expression of ACR2 in E. coli and found the inhibitory role of R84 during the anion transportation.
  • Akihiko Sakamoto, Takashi Tsukamoto, Yuji Furutani, Yuki Sudo, Kazuyuki Shimada, Akihiro Tomita, Hitoshi Kiyoi, Takashi Kato, Takashi Funatsu
    Journal of Molecular Cell Biology 8 (6) 553 - 555 1674-2788 2016/12 [Refereed][Not invited]
  • Takashi Tsukamoto, Kenji Mizutani, Taisuke Hasegawa, Megumi Takahashi, Naoya Honda, Naoki Hashimoto, Kazumi Shimono, Keitaro Yamashita, Masaki Yamamoto, Seiji Miyauchi, Shin Takagi, Shigehiko Hayashi, Takeshi Murata, Yuki Sudo
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (23) 12223 - 12232 0021-9258 2016/06 [Refereed][Not invited]
     
    Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.
  • Takashi Kikukawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Kunio Ihara, Naoki Kamo, Makoto Demura
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 1847 (8) 748 - 758 0006-3002 2015/08 [Refereed][Not invited]
  • Keiichi Inoue, Takashi Tsukamoto, Kazumi Shimono, Yuto Suzuki, Seiji Miyauchi, Shigehiko Hayashi, Hideki Kandori, Yuki Sudo
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 137 (9) 3291 - 3299 0002-7863 2015/03 [Refereed][Not invited]
     
    There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H+ flux across the membrane, raising the possibility that it works as an inward H+ pump or an H+ channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H+ transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.
  • Satoko Doi, Arisa Mori, Takashi Tsukamoto, Louisa Reissig, Kunio Ihara, Yuki Sudo
    PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES 14 (9) 1628 - 1636 1474-905X 2015 [Refereed][Not invited]
     
    Channelrhodopsins have become a focus of interest because of their ability to control neural activity by light, used in a technology called optogenetics. The channelrhodopsin in the eukaryote Chlamydomonas reinhardtii (CrChR-1) is a light-gated cation channel responsible for motility changes upon photo-illumination and a member of the membrane-embedded retinal protein family. Recent crystal structure analysis revealed that CrChR-1 has unique extended modules both at its N- and C-termini compared to other microbial retinal proteins. This study reports the first successful expression of a ChR-1 variant in Escherichia coli as a holoprotein: the ChR-1 variant lacking both the N- and C-termini (CrChR-1_82-308). However, compared to ChR-1 having the extended modules (CrChR-1_1-357), truncation of the termini greatly altered the absorption maximum and photochemical properties, including the pK(a) values of its charged residues around the chromophore, the reaction rates in the photocycle and the photo-induced ion channeling activity. The results of some experiments regarding ion transport activity suggest that CrChR-1_82-308 has a proton channeling activity even in the dark. On the basis of these results, we discuss the structural and functional roles of the N- and C-terminal extended modules in CrChR-1.
  • Takashi Tsukamoto, Makoto Demura, Yuki Sudo
    JOURNAL OF PHYSICAL CHEMISTRY B 118 (43) 12383 - 12394 1520-6106 2014/10 [Refereed][Not invited]
     
    Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane alpha-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 degrees C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 degrees C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 degrees C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.
  • Inoue K, Tsukamoto T, Sudo Y
    Biochimica et biophysica acta 1837 (5) 562 - 577 0006-3002 2014/05 [Refereed][Not invited]
  • 2014/01 [Refereed][Not invited]
  • Takashi Tsukamoto, Xianglan Li, Hiromi Morita, Takashi Minowa, Tomoyasu Aizawa, Nobutaka Hanagata, Makoto Demura
    PLOS ONE 8 (9) e75831  1932-6203 2013/09 [Refereed][Not invited]
     
    Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.
  • Takashi Tsukamoto, Keiichi Inoue, Hideki Kandori, Yuki Sudo
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (30) 21581 - 21592 0021-9258 2013/07 [Refereed][Not invited]
     
    So far retinylidene proteins (approximate to rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 degrees C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 degrees C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 x), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 x). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.
  • Takashi Tsukamoto, Takashi Kikukawa, Takuro Kurata, Kwang-Hwan Jung, Naoki Kamo, Makoto Demura
    FEBS Letters 587 (4) 322 - 327 0014-5793 2013/02/14 [Refereed][Not invited]
     
    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.
  • 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 0006-3495 2012/06 [Refereed][Not invited]
     
    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.
  • Yasutaka Yamashita, Takashi Kikukawa, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Seiji Miyauchi, Naoki Kamo, Makoto Demura
    Biochimica et Biophysica Acta (BBA) - Biomembranes 1808 (12) 2905 - 2912 0006-3002 2011/12 [Refereed][Not invited]

MISC

Presentations

  • Anion Concentration Dependency on the Photocycle of PsuACR1: Implications for the Impairment of its Fast Channel Function.  [Not invited]
    TSUKAMOTO Takashi
    18th International Conference on Retinal Proteins  2018/09
  • Substrate anion concentration significantly affects the fast channel function of Proteomonas sulcata anion channelrhodopsin-1  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第56回年会  2018/09
  • Photochemical Properties of Microbial Rhodopsin Pumps and Channels  [Invited]
    TSUKAMOTO Takashi
    APSBMS 2018 Annual Meeting  2018/07
  • 塩濃度に依存したアニオンチャネルロドプシンの光化学的性質の変化  [Not invited]
    塚本 卓
    日本生物物理学会北海道支部例会  2018/03
  • Light-driven Cl- transport mechanism of Nonlabens marinus rhodopsin-3 studied by static and time-resolved spectroscopy  [Not invited]
    塚本 卓
    日本生物物理学会第55回年会  2017/09
  • 好熱性バクテリアのレチナールタンパク質：発見と物性解析のこれまでとこれから  [Invited]
    塚本 卓
    第54回日本生化学会北海道支部例会  2017/07
  • Structural basis for high thermal stability and efficient optogenetic function of thermophilic rhodopsin  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第54回年会  2016/11
  • Cl--pumping photoreaction of a bacterial halide-ion pumping rhodopsin with an archaeal-type TSA motif  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第54回年会  2016/11
  • 微生物型ロドプシンの安定化機構の解明と光操作への展開  [Not invited]
    塚本 卓
    CREST「ファイバーレス光遺伝学による高次脳機能を支える本脳機能の解明」第１回ワークショップ  2016/11
  • 微生物型ロドプシンの光応答性と応用技術への展開  [Invited]
    塚本 卓
    新学術領域研究「人工光合成」若手育成シンポジウム〜生物から学び、応用する光反応〜  2016/11
  • 高度好熱菌Thermus thermophilus 由来サーモフィリックロドプシンの物性研究  [Not invited]
    塚本 卓
    日本生物物理学会中四国支部講演会  2016/05
  • 時間分解吸収分光法で膜タンパク質の反応中間体をとらえる  [Not invited]
    塚本 卓
    第２回創薬標的膜タンパク質の移ろいを“み（見・診・覧）る”研究会  2015/10
  • X-ray Crystal Structure of TR: Implications for High Thermal Stability and High-Performance Optogenetic Availability  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第53回年会  2015/09
  • カロテノイドを光捕集系とするレチナールタンパク質の創成と展開  [Not invited]
    塚本 卓
    新学術研究「人工光合成」第4回合同班会議  2015/06
  • Structural and Functional Characterization of Thermophilic Rhodopsin  [Not invited]
    TSUKAMOTO Takashi
    新学術研究「柔らかな分子系」第8回ワークショップ「やわらか光受容分子の理解と利用に迫るブレインストーミング研究会」  2015/01
  • 高度高熱菌由来光受容膜タンパク質ロドプシンの機能・構造研究  [Not invited]
    塚本 卓
    第１回創薬標的膜タンパク質の移ろいを“み（見・診・覧）る”研究会  2014/11
  • Physicochemical Characterization of a Light-Driven Proton Pump from an Extreme Thermophile  [Not invited]
    TSUKAMOTO Takashi
    16th International Conference on Retinal Proteins  2014/10
  • Temperature-Dependent Irreversible Structural Transition of Thermophilic Rhodopsin  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第52回年会  2014/09
  • 骨芽細胞膜タンパク質IFITM5の分子機構：翻訳後修飾，相互作用，および細胞機能  [Not invited]
    塚本 卓
    分子・物質合成プラットフォーム平成25年度シンポジウム  2014/03
  • Tsukamoto Takashi, Demura Makoto, Sudo Yuki
    Seibutsu Butsuri  2014  The Biophysical Society of Japan General Incorporated Association
  • Suzuki Kenshiro, Yamamoto Ayumi, Tsukamoto Takashi, Kobashigawa Yoshihiro, Uchida Takeshi, Inagaki Fuyuhiko, Demura Makoto, Ishimori Koichiro
    Seibutsu Butsuri  2014  The Biophysical Society of Japan General Incorporated Association
  • 生理的温度条件下における高度好熱菌由来ロドプシン・TRの物性に関する研究  [Not invited]
    塚本 卓
    分子研研究会「ロドプシン研究の故きを温ねて新しきを知る」  2013/11
  • Thermophilic rhodopsin: The first light-driven proton pump from an extreme thermophile  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第51回年会  2013/10
  • 高度好熱菌Thermus thermophilus由来微生物型ロドプシンの分光学的解析  [Not invited]
    塚本 卓
    2012年度生物物理学会中部支部講演会  2013/02
  • Suzuki Kenshiro, Yamamoto Ayumi, Tsukamoto Takashi, Kobashigawa Toshihiro, Uchida Takeshi, Inagaki Fuyuhiko, Demura Makoto, Ishimori Koichiro
    Seibutsu Butsuri  2013  The Biophysical Society of Japan General Incorporated Association
  • Mori Arisa, Tsukamoto Takashi, Yagasaki Zin, Homma Michio, Ihara Kunio, Sudo Yuki
    Seibutsu Butsuri  2013  The Biophysical Society of Japan General Incorporated Association
  • Inoue Keiichi, Tsukamoto Takashi, Yagasaki Jin, Shimono Kazumi, Miyauchi Seiji, Hayashi Shigehiko, Kandori Hideki, Sudo Yuki
    Seibutsu Butsuri  2013  The Biophysical Society of Japan General Incorporated Association
  • Tsukamoto Takashi, Sudo Yuki
    Seibutsu Butsuri  2013  The Biophysical Society of Japan General Incorporated Association
  • Kikukawa Takashi, Kusakabe Chikara, Kokubo Asami, Tsukamoto Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Ihara Kunio, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri  2013  The Biophysical Society of Japan General Incorporated Association
  • Homo-trimeric structure of Gloeobacter rhodopsin is regulated by the protonation state of Asp121, a counterion of the protonated Schiff base  [Not invited]
    TSUKAMOTO Takashi
    15th International Conference on Retinal Proteins  2012/10
  • Homotrimeric assembly of a cyanobacterial ion-pump Gloeobacter rhodopsin  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第50回年会  2012/09
  • DDM可溶化系におけるGloeobacter rhodopsinの多量体形成に関する研究  [Not invited]
    塚本 卓
    日本生物物理学会2011年度北海道支部例会  2012/03
  • Study of the oligomeric assembly of Gloeobacter rhodopsin in DDM solution  [Not invited]
    TSUKAMOTO Takashi
    9th Japan-Korea Bilateral Symposium on Biological NMR  2012/03
  • Tsukamoto Takashi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Jung Kwang-Hwan, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri  2012  The Biophysical Society of Japan General Incorporated Association
  • The role of cysteines in the first transmembrane domain of IFITM5 for the intermolecular interaction with FKBP11  [Not invited]
    TSUKAMOTO Takashi
    第34回日本分子生物学会年会  2011/12
  • Role of Ser171 for the stabilization of NpHR trimer  [Not invited]
    塚本 卓
    日本生物物理学会第49回年会  2011/09
  • Trimerization of halorhodopsin by the aromatic-interemolecular interaction and its functional modulation in detergent system  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第48回年会  2010/09
  • Trimer formation of N. pharaonis halorhodopsin is stabilized by the intermolecular interaction among Phe150 residues and leads to functional modulation in detergent solution  [Not invited]
    TSUKAMOTO Takashi
    14th International Conference on Retinal Proteins  2010/08
  • Expression, purification and characterization of osteoblast-specific membrane protein IFITM5 in detergent solubilized system  [Not invited]
    TSUKAMOTO Takashi
    第10回日本蛋白質科学会年会  2010/06
  • 膜タンパク質IFITM5の立体構造研究；溶液NMR法による立体構造解析に向けた試料調製  [Not invited]
    塚本 卓
    第48回NMR討論会  2009/11
  • ハロロドプシンNpHRの三量体構造はF150残基の対称性によって安定化される  [Not invited]
    塚本 卓
    日本生物物理学会第47回年会  2009/10
  • Structural study of IFITM5, a human double transmembrane protein; sample preparation for NMR analysis  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第47回年会  2009/10
  • Structural study of halorhodopsin: What makes its trimeric assembly stable in the DDM-solubilized system?  [Not invited]
    TSUKAMOTO Takashi
    7th Japan-Korea Bilateral Symposium on Biological NMR  2009/07
  • Molecular dynamics of photo-excited halorhodopsin in the monomer and trimer states  [Not invited]
    TSUKAMOTO Takashi
    Hokkaido University – Mahidol University Joint Symposium  2009/05
  • Molecular interaction and functional modulation of light-driven anion pump - halorhodopsin  [Not invited]
    TSUKAMOTO Takashi
    第9回日本蛋白質科学会年会  2009/05
  • Molecular dynamics of photo-excited halorhodopsin in the monomer and trimer states  [Not invited]
    TSUKAMOTO Takashi
    揺らぎと生体機能第2回公開シンポジウム  2009/03
  • Is the photoactivity of halorhodopsin modulated by the assembly of homotrimer?” 3P-097  [Not invited]
    TSUKAMOTO Takashi
    日本生物物理学会第46回年会  2008/12
  • Trimer assembly of Natronomonas pharaonis halorhodopsin, NpHR, in lipid bilayer and detergent  [Not invited]
    TSUKAMOTO Takashi
    13th International Conference on Retinal Proteins  2008/06
  • ハロロドプシン三量体の界面活性剤中での解離  [Not invited]
    塚本 卓
    日本生物物理学会第45回年会  2007/12

Teaching Experience

  • ChemistryChemistry Hokkaido University
  • SDGs and Life ScienceSDGs and Life Science Hokkaido University

Association Memberships

  • 日本蛋白質科学会   THE BIOPHYSICAL SOCIETY OF JAPAN   

Research Projects

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2023/04 -2026/03 
    Author : 塚本 卓
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 菊川 峰志, 宮内 正二, 塚本 卓, 海野 雅司
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 出村 誠, 塚本 卓
  • 天然全長型アニオンチャネルロドプシンの見過ごされてきた分子機能
    公益財団法人秋山記念生命科学振興財団：2022年度秋山記念生命科学研究助成金
    Date (from‐to) : 2022/09 -2023/03
  • トライアル研究支援制度
    北海道大学：トライアル研究支援制度
    Date (from‐to) : 2021/10 -2022/03
  • 生体分子中の窒素の特性を、中性子回折と計算化学により解明する
    日本学術振興会：科学研究費補助金
    Date (from‐to) : 2019/02 -2021/03 
    Author : 尾瀬 農之
  • イオン輸送型ロドプシンのアミノ酸変異による輸送方式の変換とその分子機構の解明
    JSPS：KAKENHI
    Date (from‐to) : 2018/04 -2020/03 
    Author : TSUKAMOTO Takashi
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : Sudo Yuki, TSUKAMOTO Takashi
     
    Retinal protein, also called "rhodopsin", has a vitamin-A aldehyde as a chromophore. It is widely distributed in the three biological domains (animals, bacteria, archaea), and is responsible for various light-dependent functions. In addition to such biological interests, recently, the new technology called "optogenetics" which is a method for controlling biological activities by light, has been established as a collaborative work with the retinal proteins. In this research, based on the background, we investigated the retinal proteins fundamentally by using various methods. Then we modified the molecular properties to develop the novel optogenetics tools that can be widely utilized for scientific research.
  • 光駆動細胞内向きプロトン輸送タンパク質の分子機構の解明と応用技術
    ノーステック財団：若手研究人材・ネットワーク育成補助金（ノースタレント補助金）
    Date (from‐to) : 2017/06 -2018/03 
    Author : 塚本 卓
  • 高度好熱菌ロドプシンにおける構造安定性の追求（研究課題番号：15K18519）
    JSPS：Grants-in-Aid for Scientific Research (KAKEN)
    Date (from‐to) : 2015/04 -2018/03 
    Author : TSUKAMOTO Takashi
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/08 -2017/03 
    Author : Kuroi Kunisato, Tsukamoto Takashi
     
    We investigated the photoreaction of the thermophilic rhodopsin (TR) by time-resolved Fourier transform infrared spectroscopy (TR-FTIR) under various temperature and pressure conditions to thermodynamically characterize its photoreaction. First, from the TR-FTIR measurement of TR and its mutants at 50 degree, we revealed the intramolecular proton transfer mechanism of TR. Second, from the TR-FTIR measurement of TR at various temperatures from 30 to 70 degrees, we found that the photoreaction mechanism of TR seemed to switch at about 40 degree. Finally, we developed the measurement system of high-pressure TR-FTIR, and applied it to the photoreaction of the bacteriorhodopsin (BR). We succeeded in measuring the TR-FTIR spectra of BR under high-pressure for the first time. However, due to the bad signal to noise ratio of the system, unfortunately we couldn’t apply that system to TR by the end of this project.
  • カロテノイド結合型ロドプシンの２光子吸収による活性化の可能性調査
    JST：Research for Specific Issues, PRESTO
    Date (from‐to) : 2016/10 -2017/03 
    Author : TSUKAMOTO Takashi
  • 時間分解赤外分光法によるレチナールタンパク質の構造ダイナミクスの解析
    自然科学研究機構分子科学研究所：平成28年度自然科学研究機構分子科学研究所共同利用研究（前期）
    Date (from‐to) : 2016/04 -2016/09 
    Author : 塚本 卓
  • 時間分解赤外分光法によるレチナールタンパク質の構造ダイナミクスの解析
    自然科学研究機構分子科学研究所：平成27年度自然科学研究機構分子科学研究所共同利用研究（後期）
    Date (from‐to) : 2015/10 -2016/03 
    Author : 塚本 卓

Social Contribution

  • Knit a Network! ロールモデル座談会
    Date (from-to) : 2021/09/16-2021/09/16
    Role : Appearance
    Sponser, Organizer, Publisher  : 北海道ダイバーシティ研究環境推進ネットワーク事務局
  • Hirameki Tokimeki Science
    Date (from-to) : 2017/08/08-2017/08/08
    Role : Lecturer
    Sponser, Organizer, Publisher  : JSPS
    Event, Program, Title : ひらめき☆ときめきサイエンス
  • 高校生限定プログラム
    Date (from-to) : 2017/08/07-2017/08/07
    Role : Lecturer
    Sponser, Organizer, Publisher  : 北海道大学
    Event, Program, Title : オープンキャンパス
  • 物理化学分野『色を感じる視覚のふしぎ』
    Date (from-to) : 2014/10/25
    Role : Lecturer
    Event, Program, Title : リケジョへの誘い 岡大方式サイエンス・トライアル
  • 中高生理系選択支援事業『理系応援キャラバン隊』
    Date (from-to) : 2007-2012
    Role : Lecturer
    Sponser, Organizer, Publisher  : 北海道大学女性研究者支援室
    Event, Program, Title : 中高生理系選択支援事業『理系応援キャラバン隊』
  • MISSION: 遺伝子コードを解読せよ！〜PCRと電気泳動で米の品種を特定する〜
    Date (from-to) : 2011
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本学術振興会
    Event, Program, Title : ひらめき☆ときめきサイエンス
  • タンパク質のイロいろ
    Date (from-to) : 2009/04
    Role : Lecturer
    Event, Program, Title : 第7回北大大学院生によるサイエンスライブ
  • 抗菌タンパク質リゾチームでバイ菌をやっつけろ！
    Date (from-to) : 2009
    Role : Lecturer
    Sponser, Organizer, Publisher  : 北海道大学
    Event, Program, Title : 北海道大学オープンキャンパス
  • 生命のミステリー 〜生命分子のかたちと働き〜『アトムの磁力でタンパク質を調べつくす！〜生物だって実は分子からできている〜』
    Date (from-to) : 2009
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本学術振興会
    Event, Program, Title : ひらめき☆ときめきサイエンス
  • タンパク質のはなし―いのちを支えるかたちとはたらき
    Date (from-to) : 2009/01
    Role : Lecturer
    Event, Program, Title : 北海道大学平成遠友夜学校
  • 抗菌タンパク質リゾチームでバイ菌をやっつけろ！
    Date (from-to) : 2008
    Role : Lecturer
    Sponser, Organizer, Publisher  : 北海道大学
    Event, Program, Title : 北海道大学オープンキャンパス
  • 生命のミステリー 〜生き物のかたちと働き〜『アトムの磁力でタンパク質を調べつくす！〜生物だって実は分子からできている〜』
    Date (from-to) : 2008
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本学術振興会
    Event, Program, Title : ひらめき☆ときめきサイエンス

Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.