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KIMURA Tetsunari
Graduate School of Science / Division of Chemistry
Associate Professor

Researcher basic information

■ Research Keyword
  • 蛋白質のダイナミクス
■ Research Areas
  • Life sciences / Biophysics
  • Nanotechnology/Materials / Biochemistry
  • Nanotechnology/Materials / Basic physical chemistry

Research activity information

■ Award
  • Oct. 2018 International Congress on Pure and Applied Chemistry Langkawi 2018, Plenary Lecture Award（International Congress on Pure and Applied Chemistry Langkawi 2018）, Membrane proteins in action; Structural and functional dynamics revealed by time-resolved measurements.
    KIMURA Tetsunari
    International society

  • Jun. 2015 Protein Science Society of Japan, 蛋白質科学会若手奨励賞, マイクロ流体フローを用いた一酸化窒素還元酵素の触媒反応の動的分光観察
    KIMURA TETSUNARI
    Japan society

■ Paper
  • Hongjie Li, Yoshiki Nakajima, Eriko Nango, Shigeki Owada, Daichi Yamada, Kana Hashimoto, Fangjia Luo, Rie Tanaka, Fusamichi Akita, Koji Kato, Jungmin Kang, Yasunori Saitoh, Shunpei Kishi, Huaxin Yu, Naoki Matsubara, Hajime Fujii, Michihiro Sugahara, Mamoru Suzuki, Tetsuya Masuda, Tetsunari Kimura, Tran Nguyen Thao, Shinichiro Yonekura, Long-Jiang Yu, Takehiko Tosha, Kensuke Tono, Yasumasa Joti, Takaki Hatsui, Makina Yabashi, Minoru Kubo, So Iwata, Hiroshi Isobe, Kizashi Yamaguchi, Michihiro Suga, Jian-Ren Shen
    Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.
    Feb. 2024, Nature, 626(7999) (7999), 670 - 677, English, International magazine
    Scientific journal

  • Hanae Takeda, Kanji Shimba, Masaki Horitani, Tetsunari Kimura, Takashi Nomura, Minoru Kubo, Yoshitsugu Shiro, Takehiko Tosha
    Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to ∼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 μs gave the NO stretching frequency at 1683 cm-1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.
    Feb. 2023, The journal of physical chemistry. B, 127(4) (4), 846 - 854, English, International magazine
    Scientific journal

  • Takashi Nomura, Tetsunari Kimura, Yusuke Kanematsu, Daichi Yamada, Keitaro Yamashita, Kunio Hirata, Go Ueno, Hironori Murakami, Tamao Hisano, Raika Yamagiwa, Hanae Takeda, Chai Gopalasingam, Ryota Kousaka, Sachiko Yanagisawa, Osami Shoji, Takashi Kumasaka, Masaki Yamamoto, Yu Takano, Hiroshi Sugimoto, Takehiko Tosha, Minoru Kubo, Yoshitsugu Shiro
    Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (I), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in I, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD+. TR-infrared (IR) spectroscopy detects the formation of I with an N–O stretching frequency of 1,290 cm−1 upon hydride transfer from NADH to the Fe3+–NO enzyme via the dissociation of NAD+ from a transient state, with an N–O stretching of 1,330 cm−1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+–NHO•− radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.
    Proceedings of the National Academy of Sciences, May 2021, Proceedings of the National Academy of Sciences, 118(21) (21), e2101481118 - e2101481118
    Scientific journal

  • Hongjie Li, Yoshiki Nakajima, Takashi Nomura, Michihiro Sugahara, Shinichiro Yonekura, Siu Kit Chan, Takanori Nakane, Takahiro Yamane, Yasufumi Umena, Mamoru Suzuki, Tetsuya Masuda, Taiki Motomura, Hisashi Naitow, Yoshinori Matsuura, Tetsunari Kimura, Kensuke Tono, Shigeki Owada, Yasumasa Joti, Rie Tanaka, Eriko Nango, Fusamichi Akita, Minoru Kubo, So Iwata, Jian-Ren Shen, Michihiro Suga
    Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -state cycle, leading to the generation of di-oxygen, protons and electrons. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.
    May 2021, IUCrJ, 8(Pt 3) (Pt 3), 431 - 443, English, International magazine
    Scientific journal

  • Kazumasa Oda, Takashi Nomura, Takanori Nakane, Keitaro Yamashita, Keiichi Inoue, Shota Ito, Johannes Vierock, Kunio Hirata, Andrés D Maturana, Kota Katayama, Tatsuya Ikuta, Itsuki Ishigami, Tamaki Izume, Rie Umeda, Ryuun Eguma, Satomi Oishi, Go Kasuya, Takafumi Kato, Tsukasa Kusakizako, Wataru Shihoya, Hiroto Shimada, Tomoyuki Takatsuji, Mizuki Takemoto, Reiya Taniguchi, Atsuhiro Tomita, Ryoki Nakamura, Masahiro Fukuda, Hirotake Miyauchi, Yongchan Lee, Eriko Nango, Rie Tanaka, Tomoyuki Tanaka, Michihiro Sugahara, Tetsunari Kimura, Tatsuro Shimamura, Takaaki Fujiwara, Yasuaki Yamanaka, Shigeki Owada, Yasumasa Joti, Kensuke Tono, Ryuichiro Ishitani, Shigehiko Hayashi, Hideki Kandori, Peter Hegemann, So Iwata, Minoru Kubo, Tomohiro Nishizawa, Osamu Nureki
    Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.
    Mar. 2021, eLife, 10, English, International magazine
    Scientific journal

  • Aliaa M Radwan, Eman F Aboelfetoh, Tetsunari Kimura, Tarek M Mohamed, Mai M El-Keiy
    BACKGROUND: Zinc oxide nanoparticles (ZnO NPs) are one of the metal oxide nanoparticles, which have attracted the interest of the researchers due to their biocompatibility, easily surface functionalization, and cancer targeting. OBJECTIVE: This study was designated to investigate the potential antitumor activity of the biologically synthesized ZnO NPs alone or in combination with doxorubicin using Ehrlich ascites carcinoma (EAC) model. METHODS: In this study, ZnO NPs were prepared by green approach using fenugreek seeds extract as reducing and capping agent then characterized by scanning electron microscope (SEM), energy dispersive x-ray (EDX), X-ray diffraction (XRD), UV-V spectroscopy, and transmission electron microscope (TEM). The prepared nanoparticles were tested for in vitro and in vivo studies using different parameters. RESULTS: Zinc oxide nanoparticles were determined to have cytotoxicity against different cancer cell lines with lower toxicity against normal one. Moreover, the in vivo study, demonstrated that the intraperitoneal injection of ZnO NPs alone or combined with doxorubicin in EAC mice inhibited the proliferation and growth of EAC by decreasing the ascetic volume and viable tumor cell count. This anti-proliferative efficiency of ZnO NPs was due to cell cycle arrest at G0/G1 phase and induction of apoptosis via upregulating the expression of caspase-3 and Bax and downregulating the expression of Bcl-2. CONCLUSION: Our findings indicated that the biologically synthesized ZnO NPs may be a promising nanomedicine therapy for cancer treatment in the future.
    Jan. 2021, Anti-cancer agents in medicinal chemistry, English, International magazine
    Scientific journal

  • Hamed A Abosharaf, Yuki Sakamoto, Aliaa M Radwan, Keisuke Yuzu, Mika Fujimura, Thoria Diab, Tarek M Mohamed, Eri Chatani, Tetsunari Kimura, Motonari Tsubaki
    Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.
    Jan. 2021, Biomolecules, 11(1) (1), English, International magazine
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Hanae Takeda, Tetsunari Kimura, Takashi Nomura, Masaki Horitani, Azusa Yokota, Akiko Matsubayashi, Shoko Ishii, Yoshitsugu Shiro, Minoru Kubo, Takehiko Tosha
    The Chemical Society of Japan, Jul. 2020, Bulletin of the Chemical Society of Japan, 93(7) (7), 825 - 833
    [Refereed]
    Scientific journal

  • Mohammed El Behery, Mika Fujimura, Tetsunari Kimura, Motonari Tsubaki
    We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of KM and Vmax. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe2+-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.
    Mar. 2020, Biochemistry and biophysics reports, 21, 100730 - 100730, English, International magazine
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Suga M, Akita F, Yamashita K, Nakajima Y, Ueno G, Li H, Yamane T, Hirata K, Umena Y, Yonekura S, Yu LJ, Murakami H, Nomura T, Kimura T, Kubo M, Baba S, Kumasaka T, Tono K, Yabashi M, Isobe H, Yamaguchi K, Yamamoto M, Ago H, Shen JR
    Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S-0 to S-4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S-1, S-2, and S-3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S-2, but flipping of D1 Glu(189) upon transition to S-3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.
    AMER ASSOC ADVANCEMENT SCIENCE, Oct. 2019, Science (New York, N.Y.), 366(6463) (6463), 334 - 338, English
    [Refereed]
    Scientific journal

  • El Behery, Mohammed, Asada, Akikazu, Takeuchi, Fusako, Kimura, Tetsunari, Chatani, Eri, TSUBAKI MOTONARI
    Chemical Society of the Philippines, Jun. 2019, KIMIKA Journal of the Kapisanang Kimika ng Pilipinas, 30(1) (1), 1 - 3, English
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Kimura, T, Lorenz-Fonfria, V.A, Douki, S, Motoki, H, Ishitani, R, Nureki, O, Higashi, M, Furutani, Y
    Magnesium ions (Mg2+) are crucial for various biological processes. A bacterial Mg2+ channel, MgtE, tightly regulates the intracellular Mg2+ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg2+ sensor. The ion selectivity for Mg2+ over Ca2+ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg2+ and Ca2+. Using site-directed mutagenesis, vibrational bands at 1421 (Mg2+), 1407 (Mg2+), ∼1440 (Ca2+), and 1390 (Ca2+) cm-1 were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg2+-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg2+ ion. The dependency of the vibrational changes on Mg2+ and Ca2+ concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg2+ ( Kd ≈ 0.3 mM) with low Ca2+ affinity ( Kd ≈ 80 mM), and a medium affinity site for Mg2+ ( Kd ≈ 2 mM) and Ca2+ ( Kd ≈ 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normal-mode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.
    Oct. 2018, J. Phys. Chem. B, 122(42) (42), 9681 - 9696, English, International magazine
    [Refereed]
    Scientific journal

  • Tetsunari KIMURA, Victor A, LORENZ-FONFRIA, Shintaro DOUKI, Hideyoshi MOTOKI, Ryuichiro ISHITANI, Osamu NUREKI, Masahiro HIGASHI, Yuji FURUTANI
    Magnesium ions (Mg2+) are crucial for various biological processes. A bacterial Mg2+ channel, MgtE, tightly regulates the intracellular Mg2+ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg2+ sensor. The ion selectivity for Mg2+ over Ca2+ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg2+ and Ca2+. Using site-directed mutagenesis, vibrational bands at 1421 (Mg2+), 1407 (Mg2+), ∼1440 (Ca2+), and 1390 (Ca2+) cm-1 were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg2+-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg2+ ion. The dependency of the vibrational changes on Mg2+ and Ca2+ concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg2+ ( Kd ≈ 0.3 mM) with low Ca2+ affinity ( Kd ≈ 80 mM), and a medium affinity site for Mg2+ ( Kd ≈ 2 mM) and Ca2+ ( Kd ≈ 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normal-mode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.
    2018, J. Phys. Chem. B., 122(42) (42), 9681 - 9696, English, International magazine
    [Refereed]
    Scientific journal

  • Ryo Komiya, Tetsunari Kimura, Takashi Nomura, Minoru Kubo, Jiwang Yan
    Single-crystal infrared (IR) spectroscopy is a promising method for protein structure analysis, where a protein crystal sample is fixed in a micro flow cell. Single-crystal calcium fluoride (CaF2) is expected as the flow cell substrate material for its excellent optical property. However, CaF2 is a highly brittle material having strong anisotropy, thus is extremely difficult to machine. Up to date, there is no available literature on fabrication of CaF2 flow cells. In this study, micro flow cells of single-crystal CaF2 were fabricated by ultraprecision cutting technology. Fly cutting was conducted using a single-crystal diamond tool having straight edges to generate a depth-varying rectangle cross section for the flow cell. The effects of cutting direction, workpiece orientation, undeformed chip thickness and tool rake angle on cutting behavior were investigated. Based on experiments and analysis, optimal conditions for ductile machining of micro grooves in CaF2 were identified. As a result, a 10 μm deep CaF2 micro flow cell with surface roughness of 2.4 nmRa was successfully fabricated. Using the fabricated flow cell, IR spectroscopic analysis of a protein single crystal at room temperature was succeeded. This study demonstrated the effectiveness of ultraprecision cutting technology in CaF2 micro flow cell fabrication, which contributes to the IR analysis of protein, and in turn, the advance of life science.
    Japan Society of Mechanical Engineers, 2018, Journal of Advanced Mechanical Design, Systems and Manufacturing, 12(1) (1), JAMDSM0021, English
    [Refereed]
    Scientific journal

  • Takehiko Tosha, Takashi Nomura, Takuma Nishida, Naoya Saeki, Kouta Okubayashi, Raika Yamagiwa, Michihiro Sugahara, Takanori Nakane, Keitaro Yamashita, Kunio Hirata, Go Ueno, Tetsunari Kimura, Tamao Hisano, Kazumasa Muramoto, Hitomi Sawai, Hanae Takeda, Eiichi Mizohata, Ayumi Yamashita, Yusuke Kanematsu, Yu Takano, Eriko Nango, Rie Tanaka, Osamu Nureki, Osami Shoji, Yuka Ikemoto, Hironori Murakami, Shigeki Owada, Kensuke Tono, Makina Yabashi, Masaki Yamamoto, Hideo Ago, So Iwata, Hiroshi Sugimoto, Yoshitsugu Shiro, Minoru Kubo
    Springer Science and Business Media LLC, Dec. 2017, Nature Communications, 8(1) (1), 1585
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Minoru Kubo, Eriko Nango, Kensuke Tono, Tetsunari Kimura, Shigeki Owada, Changyong Song, Fumitaka Mafuné, Ken Miyajima, Yoshihiro Takeda, Jun-Ya Kohno, Naoya Miyauchi, Takanori Nakane, Tomoyuki Tanaka, Takashi Nomura, Jan Davidsson, Rie Tanaka, Michio Murata, Takashi Kameshima, Takaki Hatsui, Yasumasa Joti, Richard Neutze, Makina Yabashi, So Iwata
    X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.A nanosecond pump-probe device for time-resolved serial femtosecond crystallography has been developed at SACLA.
    International Union of Crystallography, Sep. 2017, Journal of Synchrotron Radiation, 24(5) (5), 1086 - 1091, English
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Minoru Kubo, Eriko Nango, Kensuke Tono, Tetsunari Kimura, Shigeki Owada, Changyong Song, Fumitaka Mafune, Ken Miyajima, Yoshihiro Takeda, Jun-ya Kohno, Naoya Miyauchi, Takanori Nakane, Tomoyuki Tanaka, Takashi Nomura, Jan Davidsson, Rie Tanaka, Michio Murata, Takashi Kameshima, Takaki Hatsui, Yasumasa Joti, Richard Neutze, Makina Yabashi, So Iwata
    X-ray free-electron lasers (XFELs) have opened new opportunities for timeresolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TRSFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.
    INT UNION CRYSTALLOGRAPHY, Sep. 2017, JOURNAL OF SYNCHROTRON RADIATION, 24, 1086 - 1091, English
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Setsiri Haesuwannakij, Tetsunari Kimura, Yuji Furutani, Kazu Okumura, Ken Kokubo, Takao Sakata, Hidehiro Yasuda, Yumi Yakiyama, Hidehiro Sakurai
    Poly(N-vinyl-2-pyrrolidone) (PVP) of varying molecular weight (M-w = 40-360 kDa) were employed to stabilize gold nanoclusters of varying size. The resulting Au:PVP clusters were subsequently used as catalysts for a kinetic study on the sized-dependent aerobic oxidation of 1-indanol, which was monitored by time-resolved in situ infrared spectroscopy. The obtained results suggest that the catalytic behaviour is intimately correlated to the size of the clusters, which in turn depends on the molecular weight of the PVPs. The highest catalytic activity was observed for clusters with a core size of similar to 7 nm, and the size of the cluster should increase with the molecular weight of the polymer in order to maintain optimal catalytic activity. Studies on the electronic and colloid structure of these clusters revealed that the negative charge density on the cluster surface also strongly depends on the molecular weight of the stabilizing polymers.
    NATURE PUBLISHING GROUP, Aug. 2017, SCIENTIFIC REPORTS, 7(1) (1), 9579, English
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Atsuhiro Shimada, Minoru Kubo, Seiki Baba, Keitaro Yamashita, Kunio Hirata, Go Ueno, Takashi Nomura, Tetsunari Kimura, Kyoko Shinzawa-Itoh, Junpei Baba, Keita Hatano, Yuki Eto, Akari Miyamoto, Hironori Murakami, Takashi Kumasaka, Shigeki Owada, Kensuke Tono, Makina Yabashi, Yoshihiro Yamaguchi, Sachiko Yanagisawa, Miyuki Sakaguchi, Takashi Ogura, Ryo Komiya, Jiwang Yan, Eiki Yamashita, Masaki Yamamoto, Hideo Ago, Shinya Yoshikawa, Tomitake Tsukihara
    Bovine cytochrome c oxidase (CcO), a 420-kDa membrane protein, pumps protons using electrostatic repulsion between protons transferred through a water channel and net positive charges created by oxidation of heme a (Fe-a) for reduction of O-2 at heme a(3) (Fe-a3). For this process to function properly, timing is essential: The channel must be closed after collection of the protons to be pumped and before Fea oxidation. If the channel were to remain open, spontaneous backflow of the collected protons would occur. For elucidation of the channel closure mechanism, the opening of the channel, which occurs upon release of CO from CcO, is investigated by newly developed time-resolved x-ray free-electron laser and infrared techniques with nanosecond time resolution. The opening process indicates that Cu-B senses completion of proton collection and binds O-2 before binding to Fe-a3 to close the water channel using a conformational relay system, which includes Cu-B, heme a(3), and a transmembrane helix, to block backflow of the collected protons.
    AMER ASSOC ADVANCEMENT SCIENCE, Jul. 2017, SCIENCE ADVANCES, 3(7) (7), e1603042, English
    [Refereed]
    Scientific journal
    Link to Kobe Univ. Repository (Kernel)

  • Michihiro Suga, Fusamichi Akita, Michihiro Sugahara, Minoru Kubo, Yoshiki Nakajima, Takanori Nakane, Keitaro Yamashita, Yasufumi Umena, Makoto Nakabayashi, Takahiro Yamane, Takamitsu Nakano, Mamoru Suzuki, Tetsuya Masuda, Shigeyuki Inoue, Tetsunari Kimura, Takashi Nomura, Shinichiro Yonekura, Long-Jiang Yu, Tomohiro Sakamoto, Taiki Motomura, Jing-Hua Chen, Yuki Kato, Takumi Noguchi, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Takaki Hatsui, Eriko Nango, Rie Tanaka, Hisashi Naitow, Yoshinori Matsuura, Ayumi Yamashita, Masaki Yamamoto, Osamu Nureki, Makina Yabashi, Tetsuya Ishikawa, So Iwata, Jian-Ren Shen
    Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)(1-3). The structure of PSII has been analysed at 1.9 angstrom resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, `distorted-chair' form(4). This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the `radiation damage-free'(5) structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 angstrom using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 angstrom from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique mu 4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 angstrom between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously(6,7.)
    NATURE PUBLISHING GROUP, Mar. 2017, NATURE, 543(7643) (7643), 131 - +, English
    [Refereed]
    Scientific journal

  • Eriko Nango, Antoine Royant, Minoru Kubo, Takanori Nakane, Cecilia Wickstrand, Tetsunari Kimura, Tomoyuki Tanaka, Kensuke Tono, Changyong Song, Rie Tanaka, Toshi Arima, Ayumi Yamashita, Jun Kobayashi, Toshiaki Hosaka, Eiichi Mizohata, Przemyslaw Nogly, Michihiro Sugahara, Daewoong Nam, Takashi Nomura, Tatsuro Shimamura, Dohyun Im, Takaaki Fujiwara, Yasuaki Yamanaka, Byeonghyun Jeon, Tomohiro Nishizawa, Kazumasa Oda, Masahiro Fukuda, Rebecka Andersson, Petra Bath, Robert Dods, Jan Davidsson, Shigeru Matsuoka, Satoshi Kawatake, Michio Murata, Osamu Nureki, Shigeki Owada, Takashi Kameshima, Takaki Hatsui, Yasumasa Joti, Gebhard Schertler, Makina Yabashi, Ana-Nicoleta Bondar, Jorg Standfuss, Richard Neutze, So Iwata
    Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.
    AMER ASSOC ADVANCEMENT SCIENCE, Dec. 2016, SCIENCE, 354(6319) (6319), 1552 - 1557, English
    [Refereed]
    Scientific journal

  • Przemyslaw Nogly, Valerie Panneels, Garrett Nelson, Cornelius Gati, Tetsunari Kimura, Christopher Milne, Despina Milathianaki, Minoru Kubo, Wenting Wu, Chelsie Conrad, Jesse Coe, Richard Bean, Yun Zhao, Petra Bath, Robert Dods, Rajiv Harimoorthy, Kenneth R. Beyerlein, Jan Rheinberger, Daniel James, Daniel DePonte, Chufeng Li, Leonardo Sala, Garth J. Williams, Mark S. Hunter, Jason E. Koglin, Peter Berntsen, Eriko Nango, So Iwata, Henry N. Chapman, Petra Fromme, Matthias Frank, Rafael Abela, Sebastien Boutet, Anton Barty, Thomas A. White, Uwe Weierstall, John Spence, Richard Neutze, Gebhard Schertler, Jorg Standfuss
    Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
    NATURE PUBLISHING GROUP, Aug. 2016, NATURE COMMUNICATIONS, 7, 12314, English
    [Refereed]
    Scientific journal

  • Akihiro Otomo, Haruto Ishikawa, Misao Mizuno, Tetsunari Kimura, Minoru Kubo, Yoshitsugu Shiro, Shigetoshi Aono, Yasuhisa Mizutani
    CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the nu(Fe-His) mode, arising from the proximal His77 iron stretch, does not shift until 50 after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 its after the photodissociation of CO, suggesting that the nu(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 its after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.
    AMER CHEMICAL SOC, Aug. 2016, JOURNAL OF PHYSICAL CHEMISTRY B, 120(32) (32), 7836 - 7843, English
    [Refereed]
    Scientific journal

  • Miyuki Sakaguchi, Tetsunari Kimura, Takuma Nishida, Takehiko Tosha, Hiroshi Sugimoto, Yoshihiro Yamaguchi, Sachiko Yanagisawa, Go Ueno, Hironori Murakami, Hideo Ago, Masaki Yamamoto, Takashi Ogura, Yoshitsugu Shiro, Minoru Kubo
    UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2 degrees off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.
    INT UNION CRYSTALLOGRAPHY, Jan. 2016, JOURNAL OF SYNCHROTRON RADIATION, 23, 334 - 338, English
    [Refereed]
    Scientific journal

  • Ultraprecision cutting of single-crystal calcium fluoride for fabricating micro flow cell for protein structure analysis
    Ryo Komiya, Tetsunari Kimura, Minoru Kubo, Jiwang Yan
    In infrared spectroscopic analysis of the structures of protein crystals, a micro-flow cell made of single crystal calcium fluoride (CaF2) is needed. In this research, fly cutting of micro grooves on a CaF2 substrate was conducted using a single-crystal diamond tool. The effects of cutting direction, up/down cut, and tool rake angle on ductile-brittle transition of cutting behavior were investigated. Based on the experimental results and analysis, optimal cutting conditions were selected which enabled ductile machining of CaF2. As a result, a 10 μm deep micro-flow cell was successfully fabricated with surface roughness of 2.4 nmRa. The availability of the flow cell was confirmed by visible absorption spectroscopy of protein.
    Japan Society of Mechanical Engineers, Oct. 2015, Proceedings of the 8th International Conference on Leading Edge Manufacturing in 21st Century, LEM 2015, English
    International conference proceedings

  • Asumi Inaguma, Hisao Tsukamoto, Hideaki E. Kato, Tetsunari Kimura, Toru Ishizuka, Satomi Oishi, Hiromu Yawo, Osamu Nureki, Yuji Furutani
    Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, May 2015, JOURNAL OF BIOLOGICAL CHEMISTRY, 290(18) (18), 11623 - 11634, English
    [Refereed]
    Scientific journal

  • Yoshiya Inokuchi, Takayuki Ebata, Toshiaki Ikeda, Takeharu Haino, Tetsunari Kimura, Hao Guo, Yuji Furutani
    We demonstrate a powerful spectroscopic technique, surface-enhanced infrared absorption (SEIRA) spectroscopy, not only for detecting host-guest complexes in solution but also for examining the relationship between the guest selectivity, complex structure, and solvent effect. We synthesize thiol derivatives of 15-crown-5 and 18-crown-6 [2-(6-mercaptohexyloxy)methyl-15-crown-5 (15C5-C1OC6-SH) and 2-(6-mercaptohexyloxy) methyl-18-crown-6 (18C6-C1OC6-SH)], which are adsorbed on gold surfaces through S-Au bonds. The IR difference spectra of the M+center dot 15C5-C1OC6 (M = Li, Na, K, Rb, and Cs) complexes on gold are observed using aqueous solutions of MCl by SEIRA spectroscopy. The spectra show a noticeable change in the C-O stretching vibration at around 1100 cm(-1). The spectral patterns of M+center dot 15C5-C1OC6 are similar for Li+ and Na+, and for K+, Rb+, and Cs+; the interaction between the metal ions and 15C5-C1OC6 changes drastically between Na+ and K+ in the series of alkali metal ions. On the other hand, the equilibrium constant for complex formation determined by the IR intensity shows clear preference for Na+ ions. We also observe the IR difference spectra of M+center dot 18C6-C1OC6 in methanol and compare them with those in water. The spectral patterns in methanol are almost the same as those in water, but the equilibrium constant in methanol does not show preference for any ion, different from the K+ preference in water. From these findings we attribute the origin of the ion selectivity of 15C5 and 18C6 in solution to the interaction between the metal ions and the crown ethers in the complexes or the solvation energy of free ions. In the case of 15C5-C1OC6 in water, the preference of Na+ over K+, Rb+, and Cs+ can be attributed to the strength of the interaction or the size matching between metal ions and 15C5-C1OC6; the Na+ selectivity over Li+ ions is dominated by the solvation energy of free ions. For 18C6-C1OC6 in methanol, the equilibrium constant for complex formation becomes much bigger in methanol than that in water and loses the selectivity in methanol, because the solvation energy in methanol is fairly smaller than that in water, predominating the contribution from the strength of the interaction between metal ions and 18C6-C1OC6. The IR spectra measured by SEIRA spectroscopy are quite sensitive to properties of host-guest complexes such as the intermolecular interaction, the structure, and the orientation against the gold surface. However, the evidence for guest selectivity emerges primarily in the intensity of the spectra, rather than band positions or spectral patterns in the IR spectra.
    ROYAL SOC CHEMISTRY, 2015, NEW JOURNAL OF CHEMISTRY, 39(11) (11), 8673 - 8680, English
    [Refereed]
    Scientific journal

  • Yoshiya Inokuchi, Takahiro Mizuuchi, Takayuki Ebata, Toshiaki Ikeda, Takeharu Haino, Tetsunari Kimura, Hao Guo, Yuji Furutani
    We apply surface-enhanced infrared absorption (SEIRA) spectroscopy to host-guest complexes in liquid phase to examine the structural change in the complex formation. Two thiol derivatives of 18-crown-6 (18C6) are chemisorbed on a gold surface, and aqueous solutions of MCl salts (M = Li, Na, K, Rb, and Cs) are put to form M+·18C6 complexes. Infrared spectra of these complexes in the 900-2000 cm-1 region are obtained by SEIRA spectroscopy. The observed IR spectra show noticeable peaks due to the complex formation, demonstrating that SEIRA spectroscopy will be a powerful method to investigate the structure of host-guest complexes in supramolecular chemistry. © 2013 Elsevier Ltd. All rights reserved.
    Jan. 2014, Chemical Physics Letters, 592, 90 - 95, English
    [Refereed]
    Scientific journal

  • Ishii Shoko, Kimura Tetsunari, Tosha Takehiko, Shiro Yoshitsugu, Kubo Minoru
    The Biophysical Society of Japan General Incorporated Association, 2014, Seibutsu Butsuri, 54(1) (1), S264, English

  • Kimura Tetsunari, Ishii Shoko, Tosha Takehiko, Shiro Yoshitsugu, Kubo Minoru
    The Biophysical Society of Japan General Incorporated Association, 2014, Seibutsu Butsuri, 54(1) (1), S210, English

  • Hao Guo, Tetsunari Kimura, Yuji Furutani
    Surface-enhanced infrared absorption with attenuated total reflection (ATR-SEIRA) is a powerful tool for exploring molecular mechanisms of membrane proteins at the monolayer level. However, the band intensity, position, and direction can be largely influenced by the presence of a thin gold film, as observed for small molecules existing in close proximity to the surface. Here we investigated influence on the band shapes of an a-helical membrane protein, pharaonis halorhodopsin (pHR), attached on the gold surface through a complex formation between a six-histidines tag and a Ni-nitrilotriacetic acid (Ni-NTA) linker. Normal, bipolar, and inverted shapes of amide-I and -II bands were observed with an increase in film thickness, although pHR molecules would locate relatively far from the surface. The physical origin of this interesting phenomenon has been identified by changing incident angle, polarization, and film deposition rate. We find that the observed absorption anomalies are due to the influence of perpendicularly polarized light. Furthermore, it is shown that the band shapes are normal below the percolation threshold, and bipolar ones occur when an anomalous absorption by the films is strong, while the inverted ones develop with films in which surface scattering is predominant. (C) 2012 Elsevier B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Jun. 2013, CHEMICAL PHYSICS, 419, 8 - 16, English
    [Refereed]
    Scientific journal

  • Inaguma Asumi, Tsukamoto Hisao, Kimura Tetsunari, Ishizuka Toru, Yawo Hiromu, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2013, Seibutsu Butsuri, 53(1) (1), S145, English

  • Kimura Tetsunari, Tosha Takehiko, Shiro Yoshitsugu, Kubo Minoru
    The Biophysical Society of Japan General Incorporated Association, 2013, Seibutsu Butsuri, 53(1) (1), S122, English

  • Furutani Yuji, Kimura Tetsunari, Okamoto Kido
    The Biophysical Society of Japan General Incorporated Association, 2013, Seibutsu Butsuri, 53(1) (1), S142, English

  • Yuji Furutani, Tetsunari Kimura, Kido Okamoto
    Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Timeresolved measurements require a trigger, which can be controlled electronically therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding reaction of a protein. By using the step-scan mode (time resolution 2.5ms), we confirmed the completion of the buffer-exchange reaction within ~25ms the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm-1. We also demonstrated the anionbinding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm-1. It also should be noted that low sample consumption (~1 μg of protein) makes this new method a powerful technique to understand ligand-protein and ion-protein interactions, particularly for membrane proteins. ©2013 THE BIOPHYSICAL SOCIETY OF JAPAN.
    2013, Biophysics (Japan), 9, 123 - 129, English
    [Refereed]
    Scientific journal

  • Yuji Furutani, Kuniyo Fujiwara, Tetsunari Kimura, Takashi Kikukawa, Makoto Demura, Hideki Kandori
    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
    AMER CHEMICAL SOC, Oct. 2012, JOURNAL OF PHYSICAL CHEMISTRY LETTERS, 3(20) (20), 2964 - 2969, English
    [Refereed]
    Scientific journal

  • Kimura Tetsunari, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2012, Seibutsu Butsuri, 52, S24, English

  • Fujiwara Kuniyo, Kimura Tetsunari, Kikukawa Takashi, Demura Makoto, Kandori Hideki, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2012, Seibutsu Butsuri, 52, S136 - S137, English

  • Inaguma Asumi, Tsukamoto Hisao, Kimura Tetsunari, Ishizuka Toru, Yawo Hiromu, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2012, Seibutsu Butsuri, 52, S137, English

  • Xin Zhang, Vinh Q. Lam, Yun Mou, Tetsunari Kimura, Jaeyoon Chung, Sowmya Chandrasekar, Jay R. Winkler, Stephen L. Mayo, Shu-ou Shan
    Interactions between proteins underlie numerous biological functions. Theoretical work suggests that protein interactions initiate with formation of transient intermediates that subsequently relax to specific, stable complexes. However, the nature and roles of these transient intermediates have remained elusive. Here, we characterized the global structure, dynamics, and stability of a transient, on-pathway intermediate during complex assembly between the Signal Recognition Particle (SRP) and its receptor. We show that this intermediate has overlapping but distinct interaction interfaces from that of the final complex, and it is stabilized by long-range electrostatic interactions. A wide distribution of conformations is explored by the intermediate; this distribution becomes more restricted in the final complex and is further regulated by the cargo of SRP. These results suggest a funnel-shaped energy landscape for protein interactions, and they provide a framework for understanding the role of transient intermediates in protein assembly and biological regulation.
    NATL ACAD SCIENCES, Apr. 2011, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 108(16) (16), 6450 - 6455, English
    [Refereed]
    Scientific journal

  • Tsuyoshi Konuma, Tetsunari Kimura, Shuzo Matsumoto, Yuji Goto, Tetsuro Fujisawa, Alan R. Fersht, Satoshi Takahashi
    Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, Dphys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (Rg) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the Rg value extrapolated from kinetic Rg data to time zero, Rg,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in Rg2 was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated Rg of the minor component of Dphys containing the non-native cis proline isomer (Dphys,cis) to be 25.7 ± 0.6 Å. Moreover, Rg of the major component of D phys containing the native proline isomer (Dphys,tra) was estimated as 23.9 ± 0.2 Å based on Rg,0. Consequently, both components of the burst-phase intermediate of barnase (Dphys,tra and Dphys,cis) are still largely expanded. It was inferred that Dphys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase. © 2010 Elsevier Ltd. All rights reserved.
    Feb. 2011, Journal of Molecular Biology, 405(5) (5), 1284 - 1294, English
    [Refereed]
    Scientific journal

  • Furutani Yuji, Kimura Tetsunari
    The Biophysical Society of Japan General Incorporated Association, 2011, Seibutsu Butsuri, 51, S20, English

  • Muramatsu Chikako, Kawanabe Akira, Kimura Tetsunari, Kandori Hideki, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2011, Seibutsu Butsuri, 51, S103, English

  • Kimura Tetsunari, Douki Shintaro, Ishitani Ryuichiro, Nureki Osamu, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2011, Seibutsu Butsuri, 51, S109, English

  • Guo Hao, Kimura Tetsunari, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2011, Seibutsu Butsuri, 51, S108, English

  • Kimura Tetsunari, Kandori Hideki, Furutani Yuji
    The Biophysical Society of Japan General Incorporated Association, 2010, Seibutsu Butsuri, 50(2) (2), S87, English

  • Kimura Tetsunari
    The Biophysical Society of Japan General Incorporated Association, 2010, Seibutsu Butsuri, 50(2) (2), S11, English

  • Tetsunari Kimura, Akio Maeda, Shingo Nishiguchi, Koichiro Ishimori, Isao Morishima, Takashi Konno, Yuji Goto, Satoshi Takahashi
    Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mu s and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mu s and with a time constant of approximate to 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation. In contrast, I(1) possessed a feature at 1,636 cm(-1) for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I(2) possessed a line for solvated helix at 1,637 cm(-1) and major and minor lines for beta-sheet at 1,625 and 1,680 cm(-1), respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the beta-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C=O group in the beta-sheet. Together with the identification of solvated helix, the core domain of I(2) was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I(2) to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.
    NATL ACAD SCIENCES, Sep. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(36) (36), 13391 - 13396, English
    [Refereed]
    Scientific journal

  • Konuma Tsuyoshi, Kimura Tetsunari, Matsumoto Shuzo, Goto Yuji, Fujisawa Tetsuro, Takahashi Satoshi
    The Biophysical Society of Japan General Incorporated Association, 2007, Seibutsu Butsuri, 47, S132, English

  • Tetsunari Kimura, Jennifer C. Lee, Harry B. Gray, Jay R. Winkler
    The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W1W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mu s) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 mu s of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.
    NATL ACAD SCIENCES, Jan. 2007, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(1) (1), 117 - 122, English
    [Refereed]
    Scientific journal

  • T Uzawa, T Kimura, K Ishimori, Morishima, I, T Matsui, M Ikeda-Saito, S Takahashi, S Akiyama, T Fujisawa
    Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (R-g) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess R-g of 26.1(+/- 1.1) angstrom. Together with the corresponding data for proteins with different chain lengths, the seven R-g values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35 +/- 0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths. (c) 2006 Elsevier Ltd. All rights reserved.
    ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, Mar. 2006, JOURNAL OF MOLECULAR BIOLOGY, 357(3) (3), 997 - 1008, English
    [Refereed]
    Scientific journal

  • Uzawa Takanori, Kimura Tetsunari, Ishimori Koichiro, Morishima Isao, Matsui Toshitaka, Ikeda-Saito Masao, Takahashi Satoshi, Akiyama Shuji, Fujisawa Tetsuro
    The Biophysical Society of Japan General Incorporated Association, 2006, Seibutsu Butsuri, 46(2) (2), S139, English

  • T Kimura, K Sakamoto, Morishima, I, K Ishimori
    AMER CHEMICAL SOC, Jan. 2006, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(3) (3), 670 - 671, English
    [Refereed]
    Scientific journal

  • Tetsunari Kimura, Shuji Akiyama, Takanori Uzawa, Koichiro Ishimori, Isao Morishima, Tetsuro Fujisawa, Satoshi Takahashi
    Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 μs after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at ∼22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations. © 2005 Elsevier Ltd. All rights reserved.
    Jul. 2005, Journal of Molecular Biology, 350(2) (2), 349 - 362, English
    [Refereed]
    Scientific journal

  • Tetsunari Kimura, Takanori Uzawa, Koichiro Ishimori, Isao Morishima, Satoshi Takahashi, Takashi Konno, Shuji Akiyama, Tetsuro Fujisawa
    Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded β-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 μs after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding β-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape. © 2005 by The National Academy of Science of the USA.
    Feb. 2005, Proceedings of the National Academy of Sciences of the United States of America, 102(8) (8), 2748 - 2753, English
    [Refereed]
    Scientific journal

  • Takanori Uzawa, Shuji Akiyama, Tetsunari Kimura, Satoshi Takahashi, Koichiro Ishimori, Isao Morishima, Tetsuro Fujisawa
    The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (fH) and radius of gyration (R g), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to ≈50% of the overall change in Rg from the unfolded to native conformation, was observed within 300μs after the pH jump. The collapsed intermediate has a fH of 33% and a globular shape that involves > 80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
    Feb. 2004, Proceedings of the National Academy of Sciences of the United States of America, 101(5) (5), 1171 - 1176, English
    [Refereed]
    Scientific journal

  • Uzawa T., Kimura T., Takahashi S., Ishimori K., Akiyama S., Fujisawa T., Matsui T., Saito M.
    The Biophysical Society of Japan General Incorporated Association, 2004, Seibutsu Butsuri, 44, S195, Japanese

  • Kimura T., Sekiyama N., Sakamoto K., Morishima I., Ishimori K.
    The Biophysical Society of Japan General Incorporated Association, 2004, Seibutsu Butsuri, 44, S194, Japanese

  • Uzawa T., Kimura T., Takahashi S., Ishimori K., Morishima I., Akiyama S., Fujisawa T.
    The Biophysical Society of Japan General Incorporated Association, 2003, Seibutsu Butsuri, 43, S60, Japanese

  • Kinoshita M., Kimura T., Takahashi S., Ishimori K., Morishima I.
    The Biophysical Society of Japan General Incorporated Association, 2003, Seibutsu Butsuri, 43, S61, Japanese

  • Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.
    The Biophysical Society of Japan General Incorporated Association, 2003, Seibutsu Butsuri, 43, S59, Japanese

  • Shuji Akiyama, Satoshi Takahashi, Tetsunari Kimura, Koichiro Ishimori, Isao Morishima, Yukihiro Nishikawa, Tetsuro Fujisawa
    To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make smallangle x-ray scattering measurements with the time resolution of 160 μs and characterized the radius of gyration (Rg) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses ≈-20 Å of Rg, which is smaller by ≈-4 A than that of the acid-unfolded state. The Rg of the later intermediate is ≈-18 Å, which is close to that of the molten globule state. Considering the α-helix content (fH) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by Rg and fH- Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.
    Feb. 2002, Proceedings of the National Academy of Sciences of the United States of America, 99(3) (3), 1329 - 1334, English
    [Refereed]
    Scientific journal

  • Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.
    The Biophysical Society of Japan General Incorporated Association, 2002, Seibutsu Butsuri, 42(2) (2), S190, Japanese

  • Uzawa T, Akiyama S, Kimura T, Takahashi S, Ishimori K, Morishima I, Nishikawa Y, Fujisawa T
    The Biophysical Society of Japan General Incorporated Association, 2001, Seibutsu Butsuri, 41, S166, Japanese

  • Kimura T., Akiyama S., Takahashi S., Harada Y., Ishimori K., Morishima I.
    The Biophysical Society of Japan General Incorporated Association, 2000, Seibutsu Butsuri, 40, S161, Japanese

■ MISC
  • ドデシル硫酸ナトリウム共存下でのαシヌクレイン凝集反応の多様性
    北野さくら, 柚佳祐, 笹田航, 木村哲就, 茶谷絵理
    2023, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM)

  • 鉄還元機能を持つ6回膜貫通型タンパク質101F6の大腸菌での発現と機能解析
    阪口智哉, 澤井仁美, 城宜嗣, 鍔木基成, 當舎武彦, 木村哲就, 杉本宏
    2023, 日本生化学会大会(Web), 96th

  • 二液混合シリアルフェムト秒結晶構造解析による酵素反応追跡
    南後恵理子, 南後恵理子, LUO Fangjia, LUO Fangjia, 木村哲就, 菅原道泰, 中根崇智, 鈴木守, 桝田哲哉, 溝端栄一, 登野健介, 登野健介, 岩田想, 岩田想
    2021, 日本結晶学会年会講演要旨集, 2021

  • Molecular Mechanism of the Catalytic Reaction of no Reductase Revealed by Novel Time-Resolved Visible/IR Absorption Spectrometers with Microfluidic Device
    Tetsunari Kimura, Hanae Takeda, Shoko Ishii, Takehiko Tosha, Yoshitsugu Shiro, Minoru Kubo
    CELL PRESS, Feb. 2016, BIOPHYSICAL JOURNAL, 110(3) (3), 548A - 548A, English
    Summary international conference

  • SACLAにおけるシリアルフェムト秒X線結晶構造解析の技術開発
    田中智之, 菅原道泰, 中根崇智, 木村哲就, 木村哲就, 久保稔, 久保稔, 宮島謙, 宮島謙, 登野健介, 真船文隆, 真船文隆, 南後恵理子, 南後恵理子, 岩田想, 岩田想
    2016, 日本結晶学会年会講演要旨集, 2016

  • 時分割シリアルフェムト秒X線結晶解析法によるバクテリオロドプシン光反応中間体の構造解析
    南後恵理子, NEUTZE Richard, 久保稔, 久保稔, 木村哲就, 中根崇智, ROYANT Antoine, 登野健介, 岩田想
    2015, 日本結晶学会年会講演要旨集, 2015

  • 古谷 祐詞, 木村 哲就, 岡本 基土
    日本生物物理学会, 30 Sep. 2014, 生物物理, 54(5) (5), 272 - 275, Japanese
    [Refereed][Invited]
    Introduction scientific journal

  • C-2 霊長類、視覚・味覚のGPCR型受容体に対する赤外分光研究
    神取 秀樹, 片山 耕大, 古谷 祐詞, 木村 哲就
    京都大学霊長類研究所, 04 Oct. 2012, 霊長類研究所年報, 42, 118 - 118, Japanese

  • Conformational changes upon chloride-ion pumping during the pharaonis halorhodopsin photocycle studied by time-resolved FTIR difference spectroscopy
    Tetsunari Kimura, Hideki Kandori, Yuji Furutani
    AMER CHEMICAL SOC, Mar. 2011, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 241, English
    Summary international conference

  • PHYS 341-Folding dynamics of acyl-CoA binding protein probed by energy-transfer kinetics measurements
    Tetsunari Kimura, Harry B. Gray, Jay R. Winkler
    AMER CHEMICAL SOC, Aug. 2008, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 236, English
    Summary international conference

  • Direct observation of the multistep helix formation of poly-L-glutamic acids using microsecond-resolved FTIR and CD spectroscopies.
    T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I
    BIOPHYSICAL SOCIETY, Feb. 2003, BIOPHYSICAL JOURNAL, 84(2) (2), 162A - 162A, English
    Summary international conference

  • T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I
    AMER CHEMICAL SOC, Oct. 2002, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 124(39) (39), 11596 - 11597, English

■ Books And Other Publications
  • ヘムタンパク質の科学 : 生理機能の理解とその展開に向けて
    城, 宜嗣, 青野, 重利, 齋藤, 正男
    Contributor, 第１編 第５章 第５節 反応速度論, エヌ・ティー・エス, May 2022, Japanese, ISBN: 9784860437787

■ Lectures, oral presentations, etc.
  • Spectroscopic analysis of BhuUV-T reconstituted with amphiphilic polymers
    Yuki Sumida, Ayaka Naka, Yasuhiro Kobori, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    The 61st Annual Meeting of the Biophysical Society of Japa, Nov. 2023, English, 名古屋国際会議場, Domestic conference
    Poster presentation

  • Thermodynamic and kinetic analyses of interaction between aequorin and Ca2+
    Urara Kuroki, Yuka Osaki, Toshiya Funahashi, Toru Nakatsu, Tetsunari Kimura
    第96回日本生化学会大会, Nov. 2023, Japanese, 福岡国際会議場, Domestic conference
    Poster presentation

  • Investigation of the mechanism of heme transport in the ABC transporter BhuUV-T by spectroscopic methods
    Akiho Hara, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    第96回日本生化学会大会, Nov. 2023, Japanese, 福岡国際会議場, Domestic conference
    Poster presentation

  • 鉄還元機能を持つ6回膜貫通型タンパク質101F6の大腸菌での発現と機能解析
    阪口 智哉, 澤井 仁美, 城 宜嗣, 鍔木 基成, 當舎 武彦, 木村 哲就, 杉本 宏
    第96回日本生化学会大会, Nov. 2023, Japanese, 福岡国際会議場, Domestic conference
    Poster presentation

  • Development of microfluidic mixers for time-resolved SFX and spectroscopy and their applications
    Tetsunari KIMURA
    UK/Japan meeting and workshop “Dynamic Crystallography-XFELs and Synchrotrons to study enzyme reactions”, Sep. 2023, English, University of Lancaster, International conference
    [Invited]
    Oral presentation

  • Time-resolved Measurements for Structural and Functional Dynamics of Membrane Protein Complexes
    Tetsunari Kimura, Ayaka Naka, Akiho Hara, Yoshitsugu Shiro, Hiroshi Sugimoto
    The 9th International Discussion Meeting on Relaxations in Complex Systems (9 IDMRCS), Aug. 2023, English, Makuhari Messe, International conference
    [Invited]
    Oral presentation

  • Thermodynamic and kinetic analyses of chemical luminescence induced by the Ca2+ binding to Aequorin
    Urara Kuroki, Yuka Osaki, Toshiya Funahashi, Toru Nakatsu, Tetsunari Kimura
    The 23nd Annual Meeting of the Protein Science Society of Japan, Jul. 2023, English, 名古屋国際会議場, Domestic conference
    Poster presentation

  • Time-resolved spectroscopy of heme transport in ABC transporter; BhuUV-T
    Akiho Hara, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    The 23nd Annual Meeting of the Protein Science Society of Japan, Jul. 2023, English, 名古屋国際会議場, Domestic conference
    Poster presentation

  • Thermodynamic and kinetic analyses of interaction between aequorin and Ca2+
    木村哲就, 大崎優香, 黒木麗, 船橋俊也, 中津亨
    新学術領域研究「高速分子動画」領域セミナー, May 2023, English, RIKEN, Domestic conference
    Poster presentation

  • 時間分解分光測定によるタンパク質の機能・構造解析
    Tetsunari KIMURA
    東北大学多元研セミナー, Feb. 2023, Japanese, 東北大多元研, 東北大多元研, Domestic conference
    [Invited]
    Oral presentation

  • 物理化学的解析による発光タンパク質イクオリンの反応機構
    木村哲就, 大崎優香, 黒木麗, 船橋俊也, 中津亨
    新学術領域研究「高速分子動画」領域会議, Nov. 2022, Japanese, MEXT Grant-in-Aid for Scientific Research on Innovative Area ”Non-equilibrium-state molecular movies and their applications (Molecular Movies)", 淡路夢舞台, Domestic conference
    Poster presentation

  • Development and application of complementary spectroscopic methods for dynamic structural biology
    Tetsunari KIMURA
    IPR seminar, Oct. 2022, English, "Cooperation: RIKEN Pioneering Project ""Biology of Intracellular Environments"" MEXT Program for Promoting Researches on the Supercomputer Fugaku ""Biomolecular dynamics in a living cell"" MEXT Grant-in-Aid for Transformative Research Areas (A) ""New Cross Scale Biology"" MEXT Grant-in-Aid for Scientific Research on Innovative Area ”Non-equilibrium-state molecular movies and their applications (Molecular Movies)", Institute for Protein Science, Osaka Univ., International conference
    [Invited]
    Oral presentation

  • Thermodynamic analysis of the calcium binding with photoluminescence protein; aequorin
    Kuroki Urara, Funahashi Toshiya, Onishi Yusuke, Nakatsu Toru, Kimura Tetsunari
    第60回日本生物物理学会年会, Sep. 2022, English, 函館アリーナ・函館市民会館, Domestic conference
    Poster presentation

  • Conformational changes in transmembrane domain of ABC transporter revealed by double spin label-ESR spectroscopy.
    Naka Ayaka、Kobori Yasuhiro、Tsubaki Motonari、Shiro Yoshitsugu、Sugimoto Hiroshi、Kimura Tetsunari
    第60回日本生物物理学会年会, Sep. 2022, English, 函館アリーナ・函館市民会館, Domestic conference
    Poster presentation

  • Tire-resolved spectroscopic analysis of allocrite transport mechanism in heme ABC transporter; BhuUV-T
    Akiho Hara, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    第60回日本生物物理学会年会, Sep. 2022, English, 函館アリーナ・函館市民会館, Domestic conference
    Poster presentation

  • Analysis of lipid peroxidation induced by iron-reducing membrane heme protein; CYB561D2 in nanodiscs
    Aoi Yamaguchi, Motonari Tsubaki, Tetsunari Kimura
    第60回日本生物物理学会年会, Sep. 2022, English, 函館アリーナ・函館市民会館, Domestic conference
    Poster presentation

  • ITCを用いたイクオリンCa2+間相互作用の熱力学的解析
    黒木麗, 船橋俊也, 大西裕介, 中津亨, 木村哲就
    第48回生体分子科学討論会, Jun. 2022, Japanese, とりぎん文化会館小ホール, Domestic conference
    Poster presentation

  • 101F6ナノディスクによる電子移動反応の実験的解析
    Aoi Yamaguchi, Motonari Tsubaki, Tetsunari Kimura
    第48回生体分子科学討論会, Jun. 2022, Japanese, とりぎん文化会館小ホール, Domestic conference
    Poster presentation

  • 二重スピンラベル-ESR分光法を用いた ABCトランスポーターBhuUV-Tの構造解析
    仲絢香, 小堀 康博, 鍔木 基成, 城 宜嗣, 杉本 宏, 木村哲就
    第48回生体分子討論会, Jun. 2022, Japanese, とりぎん文化会館小ホール, Domestic conference
    Poster presentation

  • ABCトランスポーターBhuUV-Tにおけるヘム輸送機構の分光学的解析
    原明穂, 城宜嗣, 杉本宏, 木村哲就
    第48回生体分子科学討論会, Jun. 2022, Japanese, とりぎん文化会館小ホール, Domestic conference
    Poster presentation

  • ヘムABCトランスポーター; BhuUV-T の膜輸送に関するキネティクス解析
    Tetsunari KIMURA
    第22回日本蛋白質科学会年会, Jun. 2022, English, つくば国際会議場, Domestic conference
    [Invited]
    Oral presentation

  • Thermodynamic analysis of interaction between Aequorin and Ca2+ by ITC
    Urara Kuroki, Toshiya Funahashi, Yusuke Onishi, Toru Nakatsu, Tetsunari Kimura
    The 22nd Annual Meeting of the Protein Science Society of Japan, Jun. 2022, English, つくば国際会議場, Domestic conference
    Oral presentation

  • Spectroscopic analysis of electron transfer reaction in iron-reducing membrane protein 101F6- nanodiscs
    Aoi Yamaguchi, Motonari Tsubaki, Tetsunari Kimura
    The 22nd annual Meeting of the Protein Sience Society of Japan, Jun. 2022, English, つくば国際会議場, Domestic conference
    Oral presentation

  • ATP binding state of heme ABC transporter, BhuUV-T, investigated by double spin-labeling ESR spectroscopy
    Ayaka Naka, Yasuhiro Kobori, Motonari Tsubaki, Yositsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    the 22nd Annual Meeting og the Protein Science Society of Japan, Jun. 2022, English, つくば国際会議場, Domestic conference
    Oral presentation

  • Time-resolved spectroscopic measurements on the mechanism of heme transport by the ABC transporter BhuUV-T
    Akiho Hara, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    第22回日本蛋白質科学会年会, Jun. 2022, English, つくば国際会議場, Domestic conference
    Oral presentation

  • Microspectroscopic systemsfor time-resolved measurementsof protein microcrystals
    Tetsunari KIMURA
    新学術領域研究「高速分子動画」領域会議, May 2022, Japanese, MEXT Grant-in-Aid for Scientific Research on Innovative Area ”Non-equilibrium-state molecular movies and their applications (Molecular Movies)", RIKEN, Domestic conference
    [Invited]
    Oral presentation

  • マイクロ流路デバイスを用いた時間分解顕微分光法による膜輸送の分子機構解析
    木村 哲就
    神戸大学先端融合研究環開拓プロジェクトシンポジウム, Mar. 2022, Japanese, Online, Domestic conference
    [Invited]
    Invited oral presentation

  • 膜輸送タンパク質 ABC トランスポーターの機能・構造変化の分光学的解析
    木村 哲就
    日本分光学会 関西支部令和３年度講演会, Mar. 2022, Japanese, 日本分光学会 関西支部, Online, Domestic conference
    [Invited]
    Invited oral presentation

  • Developments and applications of time-resolved microspectroscopy for protein micro-crystals
    Tetsunari Kimura
    PacifiChem2021, Dec. 2021, English, Pacific Basin Chemical Societies, Online, International conference
    Oral presentation

  • Structural changes of a-synuclein, an intrinsically disordered protein, along the lipid-binding and oligomerization revealed by fluorescence lifetime measurements
    Ko Sasada, Tetsunari Kimura, Ryosuke Matsubara, Koichi Fujii
    PacifiChem2021, Dec. 2021, English, Pacific Basin Chemical Societies, Online, International conference
    Poster presentation

  • Transmembrane electron-transfer reaction of 101F6 protein reconstituted into nanodisc
    Aoi Yamaguchi, Hamed A. Abosharaf, Motonari Tsubaki, Tetsunari Kimura
    PacifiChem2021, Dec. 2021, English, Pacific Basin Chemical Societies, Online, International conference, Co-authored internationally
    Poster presentation

  • Spectroscopic analysis of electron transfer reaction in iron-reducing membrane protein 101F6 reconstituted into nanodiscs
    Aoi Yamaguchi, Hamed A. Abosharaf, Motonari Tsubaki, Tetsunari Kimura
    第59回日本生物物理学会年会, Nov. 2021, English, Online, Domestic conference, Co-authored internationally
    Oral presentation

  • Structural analyses of ABC transporters in nucleotide bound states investigated by CW-ESR spectroscopy
    Ayaka Naka, Yasuhiro Kobori, Motonari Tsubaki, Yoshitsugu Shiro, Hiroshi Sugimoto, Tetsunari Kimura
    第59回日本生物物理学会年会, Nov. 2021, English, Online, Domestic conference
    Oral presentation

  • Structural analysis of α-Synuclein in the lipid bilayer bound state by fluorescence lifetime measurements
    Ko Sasada, Ryosuke Matsubara, Koichi Fujii, Tetsunari Kimura
    第59回日本生物物理学会年会, Nov. 2021, English, Online, Domestic conference
    Oral presentation

  • Kinetic analysis of the transport in heme ABC transporter; BhuUV-T, by time-resolved spectroscopy
    Tetsunari Kimura
    第59回日本生物物理学会年会, Nov. 2021, English, 日本生物物理学会, Online, Domestic conference
    [Invited]
    Invited oral presentation

  • ナノディスク再構成型鉄還元膜タンパク質101F6における電子移動反応の解析
    山口葵, Abosharaf Hamed A., 鍔木基成, 木村哲就
    第94回日本生化学大会, Nov. 2021, Japanese, 日本生化学会, Online, Domestic conference, Co-authored internationally
    Poster presentation

  • Double spin-label ESR spectroscopic analysis of structural changes in heme ABC transporter induced by nucleotide-binding.
    仲絢香, 小堀康博, 鍔木基成, 城宜嗣, 杉本宏, 木村哲就
    第94回日本生化学大会, Nov. 2021, Japanese, 日本生化学会, Online, Domestic conference
    Poster presentation

  • 蛍光寿命測定によるα-Synucleinの脂質二重膜結合状態の構造解析
    笹田航, 松原亮介, 藤井宏一, 木村哲就
    第21回日本蛋白質科学会年会, Jun. 2021, Japanese, 日本蛋白質科学会, Online, Domestic conference
    Poster presentation

  • 二重スピンラベルESR分光法を用いたヘムインポーターBhuUV-Tの構造ダイナミクスの解析
    仲絢香, 小堀康博, 鍔木基成, 城宜嗣, 杉本宏, 木村哲就
    第21回日本蛋白質科学会年会, Jun. 2021, Japanese, 日本蛋白質科学会, Online, Domestic conference
    Poster presentation

  • 101F6 ナノディスクを用いた電子移動反応の実験的解析
    山口葵, Abosharaf Hamed A., 鍔木基成, 木村哲就
    第21回日本蛋白質科学会年会, Jun. 2021, Japanese, 日本蛋白質科学会, Online, Domestic conference, Co-authored internationally
    Poster presentation

  • ナノディスク再構成型BhuUV-Tによる段階的な基質輸送の分光学的観察
    木村哲就, 浅田拓也, 林沙英, 鍔木基成, 城宜嗣, 杉本宏
    第21回日本蛋白質科学会年会, Jun. 2021, Japanese, 日本蛋白質科学会, Online, Domestic conference
    Poster presentation

  • ナノディスク再構成型BhuUV-Tによる段階的な基質輸送の分光学的観察
    木村哲就, 浅田拓也, 林沙英, 鍔木基成, 城宜嗣, 杉本宏
    第21回日本蛋白質科学会年会, Jun. 2021, Japanese, 日本蛋白質科学会, Online, Domestic conference
    Oral presentation

  • ヘムABCトランスポーターBhuUVの輸送機構に関する分光学的解析
    木村哲就, 浅田拓也, 仲絢香, 林沙英, 城宜嗣, 杉本宏
    第47回生体分子科学討論会, Jun. 2021, Japanese, 第47回生体分子科学討論会実行委員会, Online, Domestic conference
    Oral presentation

  • 101F6ナノディスクを用いた電子移動反応の実験的解析
    山口葵, Abosharaf Hamed A., 鍔木基成, 木村哲就
    第47回生体分子科学討論会, Jun. 2021, Japanese, 第47回生体分子科学討論会実行委員会, Online, Domestic conference, Co-authored internationally
    Poster presentation

  • 二重スピンラベルESR分光法を用いたABCトランスポーターBhuUVの構造解析
    仲絢香, 小堀康博, 鍔木基成, 城宜嗣, 杉本宏, 木村哲就
    第47回生体分子科学討論会, Jun. 2021, Japanese, 第47回生体分子科学討論会実行委員会, Online, Domestic conference
    Poster presentation

  • α-Synucleinの脂質二重膜結合状態の分光学的構造解析
    笹田航, 松原亮介, 藤井宏一, 木村哲就
    第47回生体分子科学討論会, Jun. 2021, Japanese, 第47回生体分子科学討論会実行委員会, Online, Domestic conference
    Poster presentation

  • 分子拡散混合を利用したヘムABCトランスポーターの時間分解分光解析
    KIMURA Tetsunari
    ピコバイオロジー研究会, Dec. 2020, Japanese, 兵庫県立大ピコバイオロジー研究所, Kamigori, Domestic conference
    [Invited]
    Invited oral presentation

  • Structural and functional dynamics of ABC transporters clarified by novel time-resolved measurement systems
    KIMURA Tetsunari
    International Conference on Chemical and Environmental Sciences 2020, Dec. 2020, English, Institute of Engineering & Management, Kolkata, Online, International conference
    [Invited]
    Keynote oral presentation

  • Time-resolved spectroscopic measurements on the transport dynamics of ABC transporter
    KIMURA Tetsunari, HAYASHI Sae, IKEMOTO Yuka, SHIRO Yoshitsugu, SUGIMOTO Hiroshi
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference
    Poster presentation

  • Enhancement of ferric reductase activity of human Steap3 upon reconstitution into phospholipid bilayer nanodisc
    NISHI Ayane, NAKATA SOUTO, TAKEUCHI Fusako, KIMURA Tetsunari, TSUBAKI Motonari
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference
    Poster presentation

  • Characterization of reaction intermediate formed in the microsecond time domain of the catalytic reaction of nitric oxide reductase
    TOSHA Takehiko, TAKEDA Hanae, KIMURA Tetsunari, HORITANI Masaki, SHIRO Yoshitsugu
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference
    Poster presentation

  • Spectroscopic analysis of allocrite transport mechanism using nanodisc-reconstituted heme ABC transporter
    ASADA Takuya, TSUBAKI Motonari, SHIRO Yoshitsugu, SUGIMOTO Hiroshi, KIMURA Tetsunari
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference
    Poster presentation

  • Structural changes of a-synuclein along the lipid-binding and oligomerization revealed by fluorescence lifetime measurements.
    SASADA Ko, FUJII Koichi, MATSUBARA Ryosuke, KIMURA Tetsunari
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference
    Poster presentation

  • Could the biogenic zinc oxide nanoparticles inhibit the ATPase activity of ABC transporters?
    Aliaa M. Radwan, Mai M. El-Keiy, Tarek M. Mohamed, 木村 哲就
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference, Co-authored internationally
    Poster presentation

  • Reconistitution of Cecytb-2 in Phospholipid Bilayer Nanodisc and Meaurments of its Ferric Reductase Activity
    ABOSHARAF Hamed A., SAKAMOTO Yuki, El BEHERY Mohammed, DIAB Thoria, MOHAMED Tarek M., KIMURA Tetsunari, TSUBAKI Motonari
    第58回日本生物物理学会年会, Sep. 2020, English, The Biophysical Soceity of Japan, Online, Domestic conference, Co-authored internationally
    Poster presentation

  • 高速分子動画撮影を補完する時間分解分光測定法の開発と微結晶タンパク質試料への応用
    KIMURA Tetsunari
    第93回生化学会大会, Sep. 2020, Japanese, Japan Biochemistry Society, Online, Domestic conference
    [Invited]
    Invited oral presentation

  • Observation of Structural Change Of α-Synuclein, an Intrinsically Disordered Protein by Fluorescence Lifetime Measurement
    SASADA Ko, FUJII Koichi, MATSUBARA Ryosuke, KIMURA Tetsunari
    第20回日本蛋白質科学会, Jul. 2020, English, Protein Science Society of Japan, 誌上, Domestic conference
    Poster presentation

  • Membrane Protein Dynamics Studied by Time-resolved Measurements
    Tetsunari Kimura
    Indo-Japan Joint Workshop “Frontiers in Molecular Spectroscopy: From Fundamentals to Applications in Chemistry and Biology”, Nov. 2019, English, Kobe, International conference
    [Invited]
    Invited oral presentation

  • Complex Formation and Heme Transport of BhuUV-T Investigated by Time-Resolved UV/vis Spectroscopy
    Tetsunari Kimura
    2019 Summer Progress Report Meeting Cellular Regulation Laboratory, Univ. Hyogo, Aug. 2019, English, Univ. Hyogo, Domestic conference
    Oral presentation

  • 蛍光寿命測定によるヘリックスバンドルタンパク質ACBP の構造不均一性
    藤井 宏一, MATSUBARA RYOSUKE, TSUBAKI MOTONARI, KIMURA TETSUNARI
    第19回日本蛋白質科学会年会･第71回日本細胞生物学会大会 合同年次大会, Jun. 2019, Japanese, 神戸国際会議場, Domestic conference
    Poster presentation

  • ナノディスク再構成型ヘムABC トランスポーターを用いた基質輸送機構の分光学的解析
    浅田 拓也, TSUBAKI MOTONARI, 城 宜嗣, 杉本 宏, KIMURA TETSUNARI
    第19回日本蛋白質科学会年会･第71回日本細胞生物学会大会 合同年次大会, Jun. 2019, Japanese, 神戸国際会議場, Domestic conference
    Poster presentation

  • Elucidation of molecular functions of human tumor suppressor protein 101F6 by reconstitution into phospholipid bilayer nanodiscs
    Mohamed El-Behery, Akikazu Asada, Fusako Takeuchi, Tetsunari Kimura, Eri Chatani, ○Motonari Tsubaki
    34TH PHILIPPINE CHEMISTRY CONGRESS, May 2019, English, Waterfront Hotel and Convention Center（Philippines）, International conference
    Oral presentation

  • タンパク質の化学的ダイナミズム
    KIMURA TETSUNARI
    第9回K-CONNEX研究会, Mar. 2019, Japanese, 神戸大学六甲台第1キャンパスアカデミア館5階504 特別演習室, Domestic conference
    [Invited]
    Invited oral presentation

  • SPring-8における時間分解分光計測系の構築とタンパク質の分子機構解析への応用
    KIMURA TETSUNARI
    第38回SPring-8先端利用技術ワークショップ〜SPring-8放射光実験の生物・材料・無機化学への応用〜, Feb. 2019, Japanese, 神戸大学 六甲台キャンパス 自然科学総合研究棟1号館, Domestic conference
    [Invited]
    Invited oral presentation

  • 部位特異的スピンラベルEPR分光法によるABCトランスポーター; BhuUVの構造変化測定
    大西 萌, KOBORI YASUHIRO, 城 宜嗣, 杉本 宏, TSUBAKI MOTONARI, KIMURA TETSUNARI
    神戸大学先端融合研究環境環ワークショップ『非共有結合系の分子科学：計測技術から探る生体分子科学の新展開』, Jan. 2019, Japanese, 神戸大学 六甲台キャンパス 理学部Z201, 205号室, Domestic conference
    Poster presentation

  • 蛍光寿命計測によるACBPのフォールディング機構解析
    藤井 宏一, MATSUBARA RYOSUKE, TSUBAKI MOTONARI, KIMURA TETSUNARI
    神戸大学先端融合研究環境環ワークショップ『非共有結合系の分子科学：計測技術から探る生体分子科学の新展開』, Jan. 2019, Japanese, 神戸大学 六甲台キャンパス 理学部Z201, 204号室, Domestic conference
    Poster presentation

  • マイクロ流路ミキサーを利用したタンパク質の実時間観察
    KIMURA TETSUNARI
    神戸大学先端融合研究環境環ワークショップ『非共有結合系の分子科学：計測技術から探る生体分子科学の新展開』, Jan. 2019, Japanese, 神戸大学 六甲台キャンパス 理学部Z201, 202号室, Domestic conference
    [Invited]
    Invited oral presentation

  • Spectroscopic studies on the transport mechanism of heme ABC transporter
    林 沙英, 城 宜嗣, 杉本 宏, TSUBAKI MOTONARI, KIMURA TETSUNARI
    神戸大学先端融合研究環境環ワークショップ『非共有結合系の分子科学：計測技術から探る生体分子科学の新展開』, Jan. 2019, Japanese, 神戸大学 六甲台キャンパス 理学部Z201, 203号室, Domestic conference
    Poster presentation

  • 部位特異的スピンラベルEPR分光法によるABCトランスポーター;BhuUVの構造変化の実時間測定
    大西 萌, TSUBAKI MOTONARI, KOBORI YASUHIRO, KIMURA TETSUNARI
    神戸大学 研究基盤センター 若手フロンティア研究会2018, Dec. 2018, Japanese, 神戸大学百年記念館, Domestic conference
    Poster presentation

  • 蛍光寿命計測によるアシルCoA結合タンパク質のフォールディング機構解析
    藤井 宏一, MATSUBARA RYOSUKE, TSUBAKI MOTONARI, KIMURA TETSUNARI
    若手フロンティア研究会2018, Dec. 2018, Japanese, 神戸大学百年記念館, Domestic conference
    Poster presentation

  • 分光法による膜タンパク質のダイナミクス観察
    KIMURA TETSUNARI
    平成30 年度膜タンパク質研究会, Oct. 2018, Japanese, 淡路夢舞台国際会議場, Domestic conference
    [Invited]
    Invited oral presentation

  • Membrane proteins in action; Structural and functional dynamics revealed by time-resolved measurements.
    KIMURA TETSUNARI
    International Congress on Pure and Applied Chemistry 2018, Oct. 2018, English, Langkawi, Malaysia, International conference
    Keynote oral presentation

  • 線虫Cytochrome b561ホモログCecytb-2のアスコルビン酸由来電子伝達反応解析
    福澤 美咲, 藤村 美香, 三浦 雅央, KIMURA TETSUNARI, TSUBAKI MOTONARI
    第91回日本生化学会大会, Sep. 2018, Japanese, 国立京都国際会館, Domestic conference
    Poster presentation

  • Vibrational analysis for studying ion-protein interactions of a magnesium ion channel, MgtE
    Tetsunari Kimura, Victor Lorenz-Fonfria, Shintaro Doki, Hideyoshi Motoki, Ryuichiro Ishitani, Osamu Nureki, Masahiro Higashi, ○Yuji Furutan
    第56回日本生物物理学会年会, Sep. 2018, English, 岡山大学 津島キャンパス, Domestic conference
    Poster presentation

  • Real-time measurements of the conformational changes in ABC transporter; BhuUV, revealed by site-directed spin-labeling EPR spectroscopy
    ○Kizashi Onishi, Motonari Tsubaki, Yasuhiro Kobori, Tetsunari Kimura
    第56回日本生物物理学会年会, Sep. 2018, English, 岡山大学 津島キャンパス, Domestic conference
    Poster presentation

  • Folding dynamics of acyl-CoA binding protein revealed by fluorescence lifetime measurements
    ○Koichi Fujii, Motonari Tsubaki, Tetsunari Kimura
    第56回日本生物物理学会年会, Sep. 2018, English, 岡山大学 津島キャンパス, Domestic conference
    Poster presentation

  • Direct observation of the enzymatic reaction catalyzed by an integral membrane NO reductase using microflow-flash IR spectroscopy
    Hanae Takeda, Tetsunari Kimura, Takashi Nomura, Shoko Ishii, Akiko Matsubayashi, Azusa Yokota, Takehiko Tosha, Yoshitsugu Shiro, Minoru Kubo
    第56回日本生物物理学会年会, Sep. 2018, English, 岡山大学 津島キャンパス, Domestic conference
    Oral presentation

  • Cytochrome b561ホモログ・線虫Cecytb-2の三価鉄還元酵素活性の分子機構
    藤村 美香, 三浦 雅央, KIMURA TETSUNARI, TSUBAKI MOTONARI
    第91回日本生化学会大会, Sep. 2018, Japanese, 国立京都国際会館, Domestic conference
    Poster presentation

  • Analyses on the ascorbate-specific electron transfer function of Cecytb-2, a cytochrome b561 homolog in Caenorhabditis elegans
    ○Misaki Fukuzawa, Mika Fujimura, Masahiro Miura, Tetsunari Kimura, Motonari Tsubaki
    第56回日本生物物理学会年会, Sep. 2018, English, 岡山大学 津島キャンパス, Domestic conference
    Poster presentation

  • 線虫Cytohrome b561ホモログCecytb-2分子機能解析
    Misaki Fukuzawa, Mika Fujimra, Masahiro Miura, Tetsunari Kimura, TSUBAKI MOTONARI
    若手フロンティア研究会2017, Dec. 2017, Japanese, 神戸大学研究基盤センター、神戸大学百年記念館, Domestic conference
    Poster presentation

  • 線虫Cytochrome b561ホモログCecytb-2のアスコルビン酸からの電子伝達機能解析
    福澤 美咲, 藤村 美香, 三浦 雅央, TSUBAKI MOTONARI, KIMURA TETSUNARI
    2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, 神戸ポートアイランド, Domestic conference
    Poster presentation

  • ヘムABCトランスポーターの輸送機構に関する分光学的研究
    林 沙英, KIMURA TETSUNARI
    2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, 神戸ポートアイランド, Domestic conference
    Poster presentation

  • Cytochrome b561ホモログ・線虫Cecytb-2の分子機能解明
    藤村 美香, 三浦 雅央, KIMURA TETSUNARI, TSUBAKI MOTONARI
    2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, 神戸ポートアイランド, Domestic conference
    Poster presentation

  • ABCトランスポーターのヘム輸送機構解析
    Sae Hayashi, Hiroshi Sugimoto, Yoshitsug Shiro, Yuka Ikemoto, TSUBAKI MOTONARI, Tetsunari Kimura
    若手フロンティア研究会2017, Dec. 2017, Japanese, 神戸大学研究基盤センター、神戸大学百年記念館, Domestic conference
    Poster presentation

  • Elucidation of the molecular function of Caenorhabditis elegans Cecytb-2, a cytochrome b561 homologue
    Mika Fujimura, Masahiro Miura, KIMURA TETSUNARI, TSUBAKI MOTONARI
    第55回日本生物物理学会年会, Sep. 2017, Japanese, 熊本大学 黒髪北地区, Domestic conference
    Poster presentation

  • Direct observation of allocate-transport and ATP-hydrolysis for the ABC transport by time-resolved spectroscopy
    KIMURA TETSUNARI, HAYASHI SAE, SHIRO YOSHITSUGU, SUGIMOTO HIROSHI, IKEMOTO YUKA
    第55回日本生物物理学会年会, Sep. 2017, Japanese, 熊本大学 黒髪北地区, Domestic conference
    [Invited]
    Invited oral presentation

  • Elucidation of the NO Reduction Mechanism of Nitric Oxide Reductase Using Time-resolved Vis / IR Spectroscopy
    Hanae Takeda, KIMURA TETSUNARI, Takashi Nomura, Akiko Matsubayashi, Shoko Ishii, Takehiko Tosha, Yoshitsugu Shiro, Minoru Kubo
    第55回日本生物物理学会年会, Sep. 2017, Japanese, 熊本大学 黒髪北地区, Domestic conference
    Poster presentation

  • 線虫cytochrome b561ホモログCecytb-2の分子機能解明
    FUJIMURA MIKA, MIURA MASAHIRO, KIMURA TETSUNARI, TSUBAKI MOTONARI
    若手フロンティア研究会2016, Dec. 2016, Japanese, 神戸大学研究基盤センター, 神戸大学百年記念館、神戸、兵庫県, Domestic conference
    Poster presentation

  • Intermediate structures of the active site in membrane proteins revealed by time-resolved spectroscopy with micro-channel devices.
    Tetsunari Kimura
    シンポジウム:時空間精密構造解析による生体分子活性サイトの機能解明(第54回日本生物物理学会年会), Nov. 2016, English, つくば国際会議場, Domestic conference
    Oral presentation

  • Detection of Short-Lived Reaction Species of Nitric Oxide Reductase Using Nanoliter-Flow Time-Resolved Visible/IR Spectroscopy
    Hanae Takeda, Tetsunari Kimura, Shoko Ishii, Takehiko Tosha, Yoshitsugu Shiro, Minoru Kubo
    第54回日本生物物理学会年会, Nov. 2016, English, つくば国際会議場, Domestic conference
    Poster presentation

  • Cytochrome b561ホモログ・線虫Cecytb-2の分子機能解明
    FUJIMURA MIKA, MIURA MASAHIRO, KIMURA TETSUNARI, TSUBAKI MOTONARI
    第89回日本生化学会大会, Sep. 2016, Japanese, 宮城県、仙台国際センター、東北大学, Domestic conference
    Poster presentation

  • マイクロ流路フロー・フラッシュ時間分解赤外分光法による一酸化窒素還元酵素の反応機構解析
    KIMURA TETSUNARI, 武田英恵, 石井頌子, 當舎武彦, 城宜嗣, 久保稔
    生体分子科学討論会, Jun. 2016, Japanese, 名古屋大学 野依記念学術交流館, Domestic conference
    Oral presentation

  • NO reduction mechanism in the binuclear catalytic center of nitric oxide reductase revealed by novel micro-channel flow-flash spectrometers
    Tetsunari KIMURA, Hanae TAKEDA, Shoko ISHII, Takehiko TOSHA, Yoshitsugu SHIRO, Minoru KUBO
    Metallocofactors Gordon Research Conference, Jun. 2016, English, Stonehill College Easton, MA, International conference
    Poster presentation

  • Molecular mechanism of the catalytic reaction of NO reductase revealed by novel time-resolved visible/IR absorption spectrometers with microfluidic device.
    KIMURA Tetsunari, TAKEDA Hanae, ISHII Shoko, TOSHA Takehiko, SHIRO Yoshitsugu, KUBO Minoru
    Biophysical Soceity 60th Annual Meeting, Feb. 2016, English, Biophysical Society, Los Angeles, CA, USA, Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological reactions in the atomic and electronic level. Most of the biological reactions can be triggered by the sudden changes in buffer conditions, but the time-resolution of the conventional solution-mixing technique is limited to several milliseconds and the sample consumption i, International conference
    Poster presentation

  • Stopped-flow kinetic analysis of human duodenal cytochrome b561
    TAKEDA Hanae, TOGASHI Hiromi, KIMURA Tetsunari, Grant Mauk, SUGIMOTO Hiroshi, 城 宜嗣SHIRO Yoshitsugu
    Pacifichem2015 (The International Chemical Congress of Pacific Basin Societies 2015), Dec. 2015, English, American Chemical Society, Chemical Society of Japan, 他5カ国の化学会, Honolulu, HI, USA, International conference
    Poster presentation

  • Molecular mechanism of NO reduction by bacterial nitric oxide reductases
    SHIRO Yoshitsugu, TOSHA Takehiko, KIMURA Tetsunari, KUBO Minoru
    Pacifichem2015 (The International Chemical Congress of Pacific Basin Societies 2015), Dec. 2015, English, American Chemical Society, Chemical Society of Japan, 他5カ国の化学会, Honolulu, HI, USA, International conference
    [Invited]
    Invited oral presentation

  • Development of a time-resolved visible/IR absorption spectrometer with a novel microfluidic device and its application to catalytic reaction of NO reductase.
    KIMURA Tetsunari, ISHII Shoko, TOSHA Takehiko, SHIRO Yoshitsugu, KUBO Minoru
    Pacifichem2015 (The International Chemical Congress of Pacific Basin Societies 2015), Dec. 2015, English, American Chemical Society, Chemical Society of Japan, 他5カ国の化学会, Honolulu, HI, USA, Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological processes in molecular and atomic level, and it has been applied to observe various photocyclic reactions with the laser flash technique. However, most of the biological reactions are non-photocyclic and irreversible, and the applications of TR spectroscopy to such reactio, International conference
    Poster presentation

  • Development of Micro-channel Flow-flash Infrared Absorption Spectroscopy and its Application to Intermediate of Nitric Oxide Reductase.
    KIMURA Tetsunari, ISHII Shoko, TOSHA Takehiko, SHIRO Yoshitsugu, KUBO Minoru
    日本生物物理学会第52回年会, Sep. 2015, English, The Biophysical Soceity of Japan, Kanazawa University, Characterization of the transient intermediates in the chemical and biological reactions is essential to clarify their molecular mechanisms. In this research, we have developed an infrared spectroscopic system by combining laser spectroscopy and micro-channel flow-cell, which enabled us to monitor the irreversible enzymatic reaction of low-yield membrane protein with the nanose, Domestic conference
    Oral presentation

  • マイクロ流体フロー・フラッシュ赤外分光測定系による一酸化窒素還元酵素の触媒反応の実時間観察
    KIMURA Tetsunari, ISHII Shoko, TOSHA Takehiko, SHIRO Yoshitsugu, KUBO Minoru
    第9回分子科学討論会, Sep. 2015, Japanese, Japan Society for Molecular Science, Tokyo Institute of Technology, 酵素反応の分子機構解明には、反応過程の実時間観察が重要な役割を果たす。しかし、従来の装置では基質の結合から始まる酵素反応の動的過程においてミリ秒以内に生成する反応初期の中間体を観測することは困難である。そこで、マイクロ流体デバイスと赤外レーザーを組み合わせたフロー・フラッシュ赤外分光測定系を開発し、マイクロ秒の時間分解能での観察を可能にした。本研究では、装置を一酸化窒素還元酵素(NOR)に応用し、ヘム鉄・鉄原子から構成される活性中心においてNOがN2Oへと還元される触媒反応過程を、基質のNO伸縮振動・NN伸縮振動に着目して追跡した。当日は、速度論的解析と併せ、NORの反応機構について議論する。, Domestic conference
    Oral presentation

  • マイクロ流体フローを用いた一酸化窒素還元酵素の触媒反応の動的分光観察 (招待講演)
    KIMURA Tetsunari
    第15回日本蛋白質科学会年会, Jun. 2015, Japanese, Protein Science Society of Japan, Tokushima, 一酸化窒素還元酵素は、ヘム鉄と非ヘム鉄からなる活性中心で2分子のNOをN2Oに還元する膜蛋白質であり、細胞毒性の高いNOを速やかに無毒化する。近年、我々は結晶構造解析から、本酵素の活性中心の構造や電子・プロトン供給経路を明らかにした。一方、反応機構に関しては、これまでに、(i) 2分子のNOがヘム鉄と非ヘム鉄それぞれに結合した後、N-N間に共有結合が形成されるtrans機構と、(ii) 1分子のNOがヘム鉄と非ヘム鉄を架橋するように結合した後、2分子目のNOがNOを攻撃するcis機構が提案されており、まだ議論がなされている。これは機構解明の鍵となる過渡的中間体; NO結合型が1 ms以内に生成・消失し、従来の方法では構造解析が不可能なためである。そこで、マイクロ秒の時間分解能での計測が可能な顕微可視・赤外吸収分光装置を新規に開発し、中間体を直接観察す, Domestic conference
    [Invited]
    Invited oral presentation

  • マイクロ流体フローを用いた一酸化窒素還元酵素の触媒反応の動的分光観察 (ポスター)
    KIMURA Tetsunari
    第15回日本蛋白質科学会年会, Jun. 2015, Japanese, Protein Science Society of Japan, Tokushima, 一酸化窒素還元酵素は、ヘム鉄と非ヘム鉄からなる活性中心で2分子のNOをN2Oに還元する膜蛋白質であり、細胞毒性の高いNOを速やかに無毒化する。近年、我々は結晶構造解析から、本酵素の活性中心の構造や電子・プロトン供給経路を明らかにした。一方、反応機構に関しては、これまでに、(i) 2分子のNOがヘム鉄と非ヘム鉄それぞれに結合した後、N-N間に共有結合が形成されるtrans機構と、(ii) 1分子のNOがヘム鉄と非ヘム鉄を架橋するように結合した後、2分子目のNOがNOを攻撃するcis機構が提案されており、まだ議論がなされている。これは機構解明の鍵となる過渡的中間体; NO結合型が1 ms以内に生成・消失し、従来の方法では構造解析が不可能なためである。そこで、マイクロ秒の時間分解能での計測が可能な顕微可視・赤外吸収分光装置を新規に開発し、中間体を直接観察す, Domestic conference
    Poster presentation

  • Stopped-Flow Analysis on the Reaction of Ascorbate with Duonal Cytochrome b561
    TAKEDA Hanae, TOGASHI Hiromi, KIMURA Tetsunari, Grant Mauk, 城 宜嗣SHIRO Yoshitsugu, SUGIMOTO Hiroshi
    第15回日本蛋白質科学会年会, Jun. 2015, Japanese, Protein Science Society of Japan, Tokushima, Domestic conference
    Poster presentation

  • Preparation of Fungal NO-Reductase Crystals in the Intermediate State Using Caged-NO
    NISHIDA Takuma, TOSHA Takehiko, SAKAGUCHI Miyuki, KIMURA Tetsunari, YANAGISAWA Sachiko, UENO Tsuyoshi, MURAKAMI Hironori, YAMAMOTO Masaki, OGURA Takashi, SHIRO Yoshitsugu, KUBO Minoru
    第15回日本蛋白質科学会年会, Jun. 2015, Japanese, Protein Science Society of Japan, Tokushima, Domestic conference
    Poster presentation

■ Affiliated Academic Society
  • Protein Science Society of Japan
    2003 - Present

  • 生物物理学会（Biophysical Society, U.S.A.）
    2002 - Present

  • 日本生物物理学会
    2000 - Present

  • 日本化学会

■ Research Themes
  • 新しい動的構造解析法の開発とABCトランスポーターへの応用
    杉本 宏, 木村 哲就
    日本学術振興会, 科学研究費助成事業, 基盤研究(B), 国立研究開発法人理化学研究所, 01 Apr. 2024 - 31 Mar. 2027

  • Development of Single-Molecule Reaction Imaging Techniques for Visualizing Carbon Resource Transformation
    立川 貴士, 隈部 佳孝, 松原 亮介, 木村 哲就
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Fund for the Promotion of Joint International Research (International Collaborative Research), Kobe University, 08 Sep. 2023 - 31 Mar. 2027

  • Time-resolved measurements of allocrite transport in ABC transporters
    木村 哲就
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2022 - 31 Mar. 2025

  • 病原菌ヘムトランスポーターの構造変化の追跡と基質輸送機構の解明
    杉本 宏, 木村 哲就
    日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 国立研究開発法人理化学研究所, 01 Apr. 2021 - 31 Mar. 2024
    病原菌は宿主に感染した際に、血液ヘモグロビンから鉄をヘムの形で奪い取り、増殖に必要な栄養素として利用している。そのため、ヘムの取り込みは感染症を抑制する上でも注目されてきた。本課題では、グラム陰性の病原性細菌でアデノシン三リン酸（ATP）依存的にペリプラズム空間から細胞質へとヘムの膜輸送を行うABCトランスポーターの構造変化に着目する。これまでの当グループによる研究で明らかにした結晶構造からは、分子の中心を通る基質輸送チャネルに大きな構造変化が起こることが示唆されたが、ヘムやATPが結合した状態での構造は未だに決定できていないせいもあり、メカニズムの理解が全く進んでいない。本年度は、ヘムトランスポーターによるヘム輸送の仕組みの全容を解明するために、クライオ電子顕微鏡によって、ヘムの輸送過程の各構造のスナップショットを取得することを目指した。休止型の状態のトランスポーター（ペリプラズムヘム結合タンパク質：膜貫通サブユニット：ヌクレオチド結合サブユニットの1:2:2複合体）は比較的安定した状態であるため、試料調整方法の確立とデータ収集をおこなった。ヘム結合型の複合体の状態で界面活性剤を両親媒性ポリマーに置換することで可溶化状態を維持すると観察グリッド上での単分散性が向上することを見出した。凍結試料のクライオ電子顕微鏡観察を行なった結果、低分解能ではあるが5つのサブユニットの配置や２次構造が確認できた。

  • 時間分解構造解析を補完する精密顕微分光計測
    久保 稔, 木村 哲就, 古谷 祐詞
    日本学術振興会, 科学研究費助成事業 新学術領域研究(研究領域提案型), 新学術領域研究(研究領域提案型), 兵庫県立大学, 28 Jun. 2019 - 31 Mar. 2024
    本研究では溶液測定に加えて、タンパク質微結晶を測定できる顕微分光技術を開発し、時分割結晶構造解析（分子動画法）と時分割分光法（紫外可視吸収分光、ラマン・赤外分光、蛍光分光）との相関的・補完的解析を推進する。多くの研究試料を扱うが、それらは新学術領域「高速分子動画」において研究されている試料である。以下、各分光法毎に本年度の装置開発実績及び研究実績を記す。
    ・紫外可視吸収分光：（微結晶測定）光遺伝学ツール・チャネルロドプシンの結晶相におけるダイナミクスを解明した。その分光データと時分割結晶構造解析との相関解析により、チャネルロドプシンの光誘起チャネル開口のメカニズムを解明した。新規ロドプシン類の測定も終えており、時分割結晶構造解析との相関解析を進めている。（溶液測定）顕微装置にマイクロ流路デバイスを組み込んだ極微量フロー測定により、嫌気呼吸の鍵酵素cNORの反応中間体同定に成功した。 ・赤外分光：（微結晶測定）微結晶に適用できる顕微時分割赤外分光装置の開発を完了した。N2O発生酵素P450norに装置を適用し、反応中間体の電子状態解明に成功した。（溶液測定）DNAフォトリアーゼの反応中間体を捉えることに成功した。現在、反応中間体の化学構造決定を目指し、同位体試料の測定を試みている。 ・ラマン分光：（微結晶測定）昨年度に引き続き、時分割顕微測定系の開発を進めている。一方、凍結結晶に対する測定系を整備し、P450酵素への適用を進めている。（溶液測定）二液混合系のstopped-flow測定装置を完成させた。 ・蛍光分光：（微結晶測定）二液混合系の時分割蛍光分光装置に分光器を導入した。今後、Ca2+感受性イクオリンに適用する。（溶液測定）Stopped-flow法を用いて、イクオリンの蛍光ダイナミクスを測定した。現在、発光過程の分子モデルを構築するための速度論解析を進めている。

  • 木村 哲就
    科学研究費補助金／新学術領域研究, Apr. 2017 - Mar. 2019, Principal investigator
    Competitive research funding

  • 鍔木 基成
    学術研究助成基金助成金／基盤研究(C), Apr. 2016 - Mar. 2019
    Competitive research funding

  • Kubo Minoru, TOSHA Takehiko, KIMURA Tetsunari
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Institute of Physical and Chemical Research, 01 Apr. 2015 - 31 Mar. 2018
    This project aims to establish a time-resolved IR spectroscopic technique for studying dynamic structures of enzymes. A novel micro flow system has been developed and incorporated into a femtosecond laser-based IR spectrometer, which allows micro flow-flash IR measurements of enzymatic reactions with the minimum sample volume of 20 nL. We applied this measurement system to NO reductase, a heme enzyme that catalyzes the reduction of NO to N2O in the nitrogen cycle, and succeeded in determining the NO binding mode in the short-lived intermediate. Furthermore, we also used the micro-flow flash method for time-resolved crystallography at SACLA, and succeeded in visualizing the 3D structure of the NO-bound intermediate during the NO reduction reaction.

  • 木村 哲就
    科学研究費補助金／若手研究(A), Apr. 2015 - Mar. 2018, Principal investigator
    Competitive research funding

  • Functional expression of mammalian ion channels and analysis of the molecular mechanisms
    FURUTANI Yuji, NAKAJO Koichi, TSUKAMOTO Hisao, KIMURA Tetsunari, KUBO Yoshihiro
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, Institute for Molecular Science, 01 Apr. 2012 - 31 Mar. 2014
    Ion channel proteins work for selective permeation of ions in response to various stimuli and regulate electrical signals in living organisms. To understand the molecular mechanisms of these channels, the precise structural information with atomic resolution is required. In this study, mammalian ion channel proteins, which are important targets in medical and pharmaceutical sciences, have been studied by attenuated total reflection (ATR) FT-IR spectroscopy which gives us precise molecular information. After screening of several ion channel proteins, we have succeeded to resolve amide I bands of selectivity filter of KCNK1 channel, which is involved in regulation of resting membrane potential of cell, by using ATR FT-IR spectroscopy.

  • KIMURA Tetsunari
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A), Grant-in-Aid for Young Scientists (A), 01 Apr. 2011 - 31 Mar. 2014
    To understand the molecular mechanism of biomolecular reactions, the dynamics of the molecules along the reaction have to be revealed by the real-time measurements. However, the yield of protein is usually very low, ug-mg, and the time-resolved measurements is hard to apply for many biomolecular reactions because of the relatively large consumption. In this research, the time-resolved spectroscopies applicable to the low-yield protein samples were developed and applied to observe the folding, membrane transport and enzymatic reactions.

  • 時間分解赤外分光法を用いた膜蛋白質フォールディング機構の解明
    木村 哲就
    日本学術振興会, 科学研究費助成事業 研究活動スタート支援, 研究活動スタート支援, 分子科学研究所, 2010 - 2011
    本申請研究は、光開裂・解離性分子をβバレル膜蛋白質OmpAに修飾することで蛋白質構造に大きな摂動を加え、それによって引き起こされた変性状態からのフォールディングを生理的条件に近い温度・pH・塩濃度環境下において追跡することを目的とした。そこで、まずOmpAのβバレル構造ドメインの遺伝子配列をpETベクターに組み込んだプラスミドを作成し、そのプラスミドを形質転換した大腸菌を用いることによって発現を試みた。OmpAのβバレルドメインの発現をSDS-PAGEによって確認した。さらに簡便な精製を目的としてHisタグをC末端側に導入したプラスミドを作成すると同時に、システイン残基を導入した変異体の作製も行った。今後、光解離性分子によるOmpAの修飾を行い、構造を詳細に検討する予定である。一方、フォールディング反応に伴う膜蛋白質の構造変化に関する詳細な情報を得るためには、時間分解赤外差スペクトル分光測定を駆使する予定である。さらには、フォールディングキネティクスの解析には、得られた一連の赤外差スペクトルに対してSVD解析とグローバルフィッティング法を用いて、中間体の構造も含めて検討する必要がある。そこで、αヘリックスから構成される膜蛋白質pHRに関して、その光反応サイクルを実時間観察し、詳細な解析を行うことによって、膜蛋白質構造において起こる変化を追跡することを試みた。その結果、溶媒から隔離されたヘリックスの構造変化が光反応サイクルの後期に起こることがわかった。この予備実験により、よりドラスティックな構造変化であるフォールディング反応を時間分解赤外差スペクトル分光測定によってとらえることができると期待される。

  • マイクロ秒分割分光装置を用いた蛋白質の折り畳み機構の解明
    基礎科学特別研究員研究費, 2003 - 2005
    Competitive research funding

  • 圧力効果を利用した蛋白質の折り畳み機構における水分子ダイナミクスの直接観察
    基礎科学特別研究員研究費, 2005
    Competitive research funding

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