Structural analyses of reaction states of ion-pumping rhodopsin

• Project/Area Number: 19K06582
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: Nagoya University
• Principal Investigator: Kouyama Tsutomu 名古屋大学, 理学研究科, 名誉教授 (30170210)
• Co-Investigator(Kenkyū-buntansha): 井原 邦夫 名古屋大学, 遺伝子実験施設, 准教授 (90223297)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: レチナール蛋白質 / 光駆動プロトンポンプ / 光駆動塩素イオンポンプ / レチナールタンパク質 / 生体イオン輸送 / バクテリオロドプシン / アーキロドプシン / ハロロドプシン / 反応中間体の構造解析 / 光反応サイクル / 生体イオンポンプ / 光駆動陰イオンポンプ / レチナール光異性化 / X線結晶構造解析 / 光駆動イオンポンプ / ロドプシン

Outline of Research at the Start

申請者は光駆動塩素イオンポンプ・ハロロドプシンの反応状態のX線構造解析を行い、イオン輸送サイクルにおいて発色団レチナールが3種類の異性化状態を経ることを明らかにするなど、イオン輸送機構の理解に貢献してきた。一方、光駆動プロトンポンプ・バクテリオロドプシンに関しては、いくつかの反応中間体の構造決定を行ってきたものの、光サイクルの後半部分で起こる事象については未解明のままである。本研究課題においては、別種の光駆動プロトンポンプを用いて、プロトン輸送サイクルの後半で生じる反応中間体の構造決定を企て、プロトン輸送サイクルにおいてもレチナールの3段階の異性化反応が起きていることを検証する。

Outline of Final Research Achievements

In bacteriorhodopsin (bR), the proton pumping cycle is initiated by the photo-isomerization of the retinal chromophore with the 13-trans configuration into the 13-cis configuration, and thermal re-isomerization into the initial trans configuration occurs in the final step of the reaction cycle. We have shown that the O intermediate formed during the photo cycle has two substates (O1 and O2) with different spectral characteristics, and thermally activated isomerization of retinal into the all-trans configurotion occurs at the O1→O2 transition. We also observed that the blue all-trans isomer at acidic pH has similar spectroscopic properties to the O2 intermediate, and discussed the possibility that that the mechanism of the retinal re-isomerization taking place in the late stage of the proton pumping cycle is analogous to that of thermal isomerization during dark adaptation in bR, which involves red shifted intermediates (isomers of blue membrane).

Academic Significance and Societal Importance of the Research Achievements

バクテリオロドプシン（BR)の発見以来、たくさんの微生物型ロドプシンが古細菌、真正細菌、真核生物に見出されており、それらのいくつかはニューロン活動を光制御するのに利用されている。新しく発見された微生物ロドプシンの機能・構造が精力的に調べられているが、多くの場合、微生物ロドプシンの反応スキームはBRの従来の反応スキーム（＝レチナール再異性化の記述が曖昧）を参考にして組み上げられている。本研究ではO中間体には2種類のサブ状態（O1とO2）が存在し、O1→O2 転移でレチナールの再異性化が起こることが示された。我々が提唱した反応スキームは、より強力な光遺伝学的ツールを設計するのに役立つであろう。

Research Products

• [Journal Article] Structural mechanism of microbial rhodopsins: Report for the session 4 at the 19^th International Conference on Retinal Proteins (2023)
  • Author(s): Kouyama Tsutomu、Dencher Norbert A.
  • Journal Title: Biophysics and Physicobiology Volume: 20 Issue: Supplemental Pages: n/a
  • DOI: 10.2142/biophysico.bppb-v20.s014
  • ISSN: 2189-4779
  • Peer Reviewed
• [Journal Article] Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12 (2023)
  • Author(s): Tanudjaja Ellen、Hoshi Naomi、Yamamoto Kaneyoshi、Ihara Kunio、Furuta Tadaomi、Tsujii Masaru、Ishimaru Yasuhiro、Uozumi Nobuyuki
  • Journal Title: Journal of Biological Chemistry Volume: 299 Issue: 2 Pages: 102846-102846
  • DOI: 10.1016/j.jbc.2022.102846
  • Peer Reviewed / Open Access
• [Journal Article] Existence of two substates in the O intermediate of the bacteriorhodopsin photocycle (2022)
  • Author(s): Kouyama Tsutomu、Ihara Kunio
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Biomembranes Volume: 1864 Issue: 10 Pages: 183998-183998
  • DOI: 10.1016/j.bbamem.2022.183998
  • Peer Reviewed
• [Journal Article] Two substates in the O intermediate of the light-driven proton pump archaerhodopsin-2 (2022)
  • Author(s): Kouyama Tsutomu、Ihara Kunio
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Biomembranes Volume: 1864 Issue: 7 Pages: 183919-183919
  • DOI: 10.1016/j.bbamem.2022.183919
  • Peer Reviewed
• [Journal Article] Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor (2022)
  • Author(s): Terashima Hiroyuki、Hori Kiyoshiro、Ihara Kunio、Homma Michio、Kojima Seiji
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 2979-2979
  • DOI: 10.1038/s41598-022-06947-5
  • Peer Reviewed / Open Access
• [Journal Article] Identification of <i>ksg1</i> mutation showing long‐lived phenotype in fission yeast (2021)
  • Author(s): Matsui Kotaro、Okamoto Keisuke、Hasegawa Tomoka、Ohtsuka Hokuto、Shimasaki Takafumi、Ihara Kunio、Goto Yuhei、Aoki Kazuhiro、Aiba Hirofumi
  • Journal Title: Genes to Cells Volume: 26 Issue: 12 Pages: 967-978
  • DOI: 10.1111/gtc.12897
  • Peer Reviewed / Open Access
• [Journal Article] A slight bending of an α-helix in FliM creates a counterclockwise-locked structure of the flagellar motor in <i>Vibrio</i> (2021)
  • Author(s): Takekawa Norihiro、Nishikino Tatsuro、Yamashita Toshiki、Hori Kiyoshiro、Onoue Yasuhiro、Ihara Kunio、Kojima Seiji、Homma Michio、Imada Katsumi
  • Journal Title: The Journal of Biochemistry Volume: 170 Issue: 4 Pages: 531-538
  • DOI: 10.1093/jb/mvab074
  • Peer Reviewed / Open Access
• [Journal Article] Identification of <i>sur2</i> mutation affecting the lifespan of fission yeast (2021)
  • Author(s): Kurauchi Tatsuhiro、Matsui Kotaro、Shimasaki Takafumi、Ohtsuka Hokuto、Tsubouchi Satoshi、Ihara Kunio、Tani Motohiro、Aiba Hirofumi
  • Journal Title: FEMS Microbiology Letters Volume: 368 Issue: 12
  • DOI: 10.1093/femsle/fnab070
  • NAID: 120007183219
  • Peer Reviewed
• [Journal Article] Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli (2021)
  • Author(s): Sugawara Keita、Toyoda Hayato、Kimura Mami、Hayasaka Shunsuke、Saito Hiromi、Kobayashi Hiroshi、Ihara Kunio、Ida Tomoaki、Akaike Takaaki、Ando Eiji、Hyodo Mamoru、Hayakawa Yoshihiro、Hamamoto Shin、Uozumi Nobuyuki
  • Journal Title: Biochemical Journal Volume: 478 Issue: 1 Pages: 41-59
  • DOI: 10.1042/bcj20200723
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Presynaptic MAST kinase controls opposing postsynaptic responses to convey stimulus valence in Caenorhabditis elegans (2020)
  • Author(s): Nakano Shunji、Ikeda Muneki、Tsukada Yuki、Fei Xianfeng、Suzuki Takamasa、Niino Yusuke、Ahluwalia Rhea、Sano Ayana、Kondo Rumi、Ihara Kunio、Miyawaki Atsushi、Hashimoto Koichi、Higashiyama Tetsuya、Mori Ikue
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 117 Issue: 3 Pages: 1638-1647
  • DOI: 10.1073/pnas.1909240117
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach. (2020)
  • Author(s): Mizukami T, Furuzawa S, Itoh SG, Segawa S, Ikura T, Ihara K, Okumura H, Roder H, Maki K
  • Journal Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 117 Issue: 33 Pages: 19953-19962
  • DOI: 10.1073/pnas.1914349117
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii (2020)
  • Author(s): Matsuo Takuya, Iida Takahiro, Ohmura Ayumi, Gururaj Malavika, Kato Daisaku, Mutoh Risa, Ihara Kunio, Ishiura Masahiro
  • Journal Title: PLOS GENETICS Volume: 16 Issue: 6 Pages: e1008814-e1008814
  • DOI: 10.1371/journal.pgen.1008814
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt (2020)
  • Author(s): Enomoto Shigeaki, Shimane Yasuhiro, Ihara Kunio, Kamekura Masahiro, Itoh Takashi, Ohkuma Moriya, Takahashi--Ando Naoko, Fukushima Yasumasa, Yoshida Yasuhiko, Usami Ron, Takai Ken, Minegishi Hiroaki
  • Journal Title: INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY Volume: 70 Issue: 12 Pages: 6331-6337
  • DOI: 10.1099/ijsem.0.004535
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The mechanosensitive channel YbdG from Escherichia coli has a role in adaptation to osmotic up-shock. (2019)
  • Author(s): Shun Amemiya, Hayato Toyoda, Mami Kimura, Hiromi Saito, Hiroshi Kobayashi, Kunio Ihara 3 , Kiyoto Kamagata 4 , Ryuji Kawabata 5 , Setsu Kato, Yutaka Nakashimada, Tadaomi Furuta, Shin Hamamoto, Nobuyuki Uozumi
  • Journal Title: Journal of Biological Chemistry Volume: 294 (33) Issue: 33 Pages: 12281-12292
  • DOI: 10.1074/jbc.ra118.007340
  • Peer Reviewed
• [Journal Article] gas1 mutation extends chronological lifespan via Pmk1 and Sty1 MAPKs in Schizosaccharomyces pombe (2019)
  • Author(s): Imai Yuki、Shimasaki Takafumi、Enokimura Chihiro、Ohtsuka Hokuto、Tsubouchi Satoshi、Ihara Kunio、Aiba Hirofumi
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 84 Issue: 2 Pages: 330-337
  • DOI: 10.1080/09168451.2019.1676695
  • Peer Reviewed
• [Presentation] Existence of two substates in the O intermediate of the bacteriorhodopsin photocycle (2021)
  • Author(s): Kouyama T, Ihara K.
  • Organizer: 日本生物物理学会年会
• [Presentation] バクテリオロドプシンおよびアーキロドプシンのプロトン輸送サイクルにおける2種類のO中間体の出現 (2021)
  • Author(s): 神山勉、井原邦夫
  • Organizer: 日本生物物理学会中部支部講演会
• [Presentation] 筋収縮と能動輸送 (2020)
  • Author(s): 神山勉
  • Organizer: 日本物理学会