Understanding the mechanism of structural stability for thermophilic rhodopsin

• Project/Area Number: 15K18519
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Hokkaido University (2017); Okayama University (2015-2016)
• Principal Investigator: Tsukamoto Takashi 北海道大学, 先端生命科学研究院, 助教 (30744271)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2017: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: ロドプシン / 膜タンパク質 / イオンポンプ / 構造安定性 / 高度好熱菌 / フラッシュフォトリシス / プロトン移動 / 光受容 / 熱安定性 / 構造安定化 / 光遺伝学

Outline of Final Research Achievements

This research was carried out for a photoreceptor protein called thermophilic rhodopsin (abbreviated as TR), which we have discovered from a thermophilic bacterium for the first time in 2013, (1) to reveal the structural factor for the high thermal stability of TR, and (2) to evaluate the structural stability against some physical and chemical stimuli other than heat. For the purpose (1), it was revealed that TR was highly stabilized by hydrophobic interactions inside the protein by combining the analyses of UV-Vis absorption spectra, X-ray crystallography, and molecular dynamics simulation. For the purpose (2), the exhausted evaluation for the structural stability by using some chemical reagents revealed the correlation between the mechanism for stabilizing the TR structure against heat and that against some chemical stimuli.

Research Products

• [Int'l Joint Research] Weizmann Institute of Science/The Hebrew University of Jerusalem(Israel)
• [Journal Article] Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism (2018)
  • Author(s): Inoue Saki、Yoshizawa Susumu、Nakajima Yu、Kojima Keiichi、Tsukamoto Takashi、Kikukawa Takashi、Sudo Yuki
  • Journal Title: Phys. Chem. Chem. Phys. Volume: 20 Issue: 5 Pages: 3172-3183
  • DOI: 10.1039/c7cp05033j
  • Peer Reviewed
• [Journal Article] Presence of a Haloarchaeal Halorhodopsin-Like Cl⁻ Pump in Marine Bacteria (2018)
  • Author(s): Nakajima Yu、Tsukamoto Takashi、Kumagai Yohei、Ogura Yoshitoshi、Hayashi Tetsuya、Song Jaeho、Kikukawa Takashi、Demura Makoto、Kogure Kazuhiro、Sudo Yuki、Yoshizawa Susumu
  • Journal Title: Microbes and Environments Volume: 33 Issue: 1 Pages: 89-97
  • DOI: 10.1264/jsme2.ME17197
  • NAID: 130006581777
  • ISSN: 1342-6311, 1347-4405
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Comparative evaluation of the stability of seven-transmembrane microbial rhodopsins to various physicochemical stimuli (2017)
  • Author(s): Honda Naoya、Tsukamoto Takashi、Sudo Yuki
  • Journal Title: Chem. Phys. Lett. Volume: 682 Pages: 6-14
  • DOI: 10.1016/j.cplett.2017.05.055
  • Peer Reviewed
• [Journal Article] A phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941T (2017)
  • Author(s): Kanehara K, Yoshizawa S, Tsukamoto T, *Sudo Y.
  • Journal Title: Sci. Rep. Volume: 7 Issue: 1 Pages: 44427-44427
  • DOI: 10.1038/srep44427
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics (2017)
  • Author(s): Tsukamoto, T., Yoshizawa, S., Kikukawa, T., Demura, M., Sudo, Y.
  • Journal Title: The Journal of Physical Chemistry B Volume: 121 Pages: 2027-2038
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Demonstration of a light-driven SO42- transporter and its spectroscopic characteristics (2017)
  • Author(s): Niho A, Yoshizawa S, Tsukamoto T, Kurihara M, Tahara S, Nakajima Y, Mizuno M, Kuramochi H, Tahara T, Mizutani Y, *Sudo Y.
  • Journal Title: J. Am. Chem. Soc. Volume: 139 Issue: 12 Pages: 4376-4389
  • DOI: 10.1021/jacs.6b12139
  • NAID: 120006425837
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli (2017)
  • Author(s): Doi S, Tsukamoto T, Yoshizawa S, & *Sudo Y.
  • Journal Title: Sci. Rep. Volume: 7 Issue: 1 Pages: 41879-41879
  • DOI: 10.1038/srep41879
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] X-ray crystallographic structure of thermophilic rhodopsin: implications for high thermal stability and optogenetic function (2016)
  • Author(s): Tsukamoto T, Mizutani K, Hasegawa T, Takahashi M, Honda N, Hashimoto N, Shimono K, Yamashita K, Yamamoto M, Miyauchi S, Takagi S, Hayashi S, Murata T, Sudo Y.
  • Journal Title: J. Biol. Chem. Volume: in press Issue: 23 Pages: 12223-12232
  • DOI: 10.1074/jbc.m116.719815
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 塩濃度に依存したアニオンチャネルロドプシンの光化学的性質の変化 (2018)
  • Author(s): 塚本卓, 菊地ちひろ, 鈴木拓, 相沢智康, 菊川峰志, 出村誠
  • Organizer: 日本生物物理学会北海道支部例会
• [Presentation] Na+ポンプ型ロドプシンの機能解析：光誘起Na+濃度変化の直接測定の試み (2018)
  • Author(s): 村部圭佑, 菊川峰志, 塚本卓, 相沢智康, 出村誠
  • Organizer: 日本生物物理学会北海道支部例会
• [Presentation] Large-scale expression of anti-microbial peptide cecropin P1 (CP1) in E. coli using calmodulin as fusion partner(抗菌ペプチドcecropin P1 (CP1)のcalmodulinを用いたE. coliを宿主とした大量発現) (2018)
  • Author(s): 加藤貴純, 石田博昭, 大沼幸暉, 塚本卓, 菊川峰志, 出村誠, ホーゲル ハンス, 相沢智康
  • Organizer: 日本農芸化学会2018
• [Presentation] NMRメタボロミクスに適した生体試料調製法の検討 (2017)
  • Author(s): 山野めぐみ，稲村勇雅，北田直也, 清水由宇，久米田博之，熊木康裕，菊川峰志，塚本卓，出村誠，中村公則，綾部時芳，相沢智康
  • Organizer: 第５６回 NMR討論会
• [Presentation] NMRメタボローム解析を用いたルーメン内繊維分解細菌間相互作用に関する研究 (2017)
  • Author(s): 稲村 勇雅，山野 めぐみ，北田 直也，久米田 博之，熊木 康裕，大西 裕季，塚本 卓，菊川 峰志，出村 誠，福間 直希，村住 侑毅，小池 聡，小林 康男，相沢 智康
  • Organizer: 第５６回 NMR討論会
• [Presentation] 好熱菌由来光駆動プロトンポンプの発色団構造および多量体安定性に膜環境が与える効果 (2017)
  • Author(s): 塩谷智巳、水野操、塚本卓、須藤雄気、水谷泰久
  • Organizer: 日本化学会第97春季大会
• [Presentation] 好熱性バクテリアのレチナールタンパク質：発見と物性解析のこれまでとこれから (2017)
  • Author(s): 塚本卓
  • Organizer: 第54回日本生化学会北海道支部例会
  • Invited
• [Presentation] Ultrasensitive neural silencing activity of anion channelrhodopsin-2 (ACR2) in Caenorhabditis elegans (2017)
  • Author(s): Maki, M., Doi, S., Kojima, K., Chowdhury, S., Tsukamoto, T., Yamanaka, A., Takagi, S., Sudo, Y.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Functional analysis of T126E mutant of Natronomonas pharaonis halorhodopsin (2017)
  • Author(s): Abe, S., Kikukawa, T., Tsukamoto, T., Aizawa, T., Kamo, N., Demura, M.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Functional analysis of Na+-pumping rhodopsin focusing on acidic residues on the extracellular surface (2017)
  • Author(s): Okamura, A., Kikukawa, T., Tsukamoto, T., Aizawa, T., Kamo, N., Demura, M.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Over expression of recombinant peamaclein, a cycteine-rich plant allergenic peptide derived from peach pulp (2017)
  • Author(s): Suzuki, H., Kato, T., Yasumoto, M., Iwama, K., Okamura, A., Kato, T., Kitada, N., Kuddus, Md. R., Rumi, F., Tsukamoto, T., Kikukawa, T., Demura, M., Aizawa, T.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Functional importance of trimer formation of light-driven H+ pump Gloeobacter rhodoosin (2017)
  • Author(s): Iizuka, A., Kikukawa, T., Kajimoto, K., Fujisawa, T., Tsukamoto, T., Aizawa, T., Kamo, N., Unno, M., Demura, M.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Analysis of Na+ transfer reactions of Na+-pumping rhodopsin (2017)
  • Author(s): Murabe, K., Kikukawa, T., Tsukamoto, T., Aizawa, T., Kamo, N., Demura, M.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Light-driven Cl- transport mechanism of Nonlabens marinus rhodopsin-3 studied by static and time-resolved spectroscopy (2017)
  • Author(s): Tsukamoto, T., Yoshizawa, S., Kikukawa, T., Demura, M.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Mass production of fowlicidin, a cathelicidin antimicrobial peptide by a calmodulin-peptide fusion and coexpression system (2017)
  • Author(s): Onuma, K., Ishida, H., Kato, T., Tsukamoto, T., Kikukawa, T., Demura, M., Vogel, HJ., Aizawa, T.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Spectroscopic analysis of RmXeR, an inward H+ pump rhodopsin from the marine bacterium Rubricoccus marinus SG-29T (2017)
  • Author(s): Inoue, S., Yoshizawa, S., Kojima, K., Tsukamoto, T., Kikukawa, T., Sudo, Y.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] Distinctive functional and photochemical properties among light-driven sodium ion pumping rhodopsins (2017)
  • Author(s): Kurihara, M., Hashimoto, M., Yoshizawa, S., Kojima, K., Tsukamoto, T., Kikukawa, T., Sudo, Y.
  • Organizer: 日本生物物理学会第55回年会
• [Presentation] 好熱菌由来光駆動プロトンポンプTRのタンパク質環境が発色団構造に及ぼす影響 (2017)
  • Author(s): 塩谷智巳、水野操、塚本卓、須藤雄気、水谷泰久
  • Organizer: 第11回分子科学討論会
• [Presentation] Comparative studies on light-driven sodium pumping rhodopsins (2017)
  • Author(s): Kurihara, M., Hashimoto, M., Yoshizawa, S., Kojima, K., Tsukamoto, T., Kikukawa, T., Sudo, Y.
  • Organizer: 日本生物物理学会中四国支部講演会
• [Presentation] Investigation of a phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941T (2017)
  • Author(s): Kanehara, K., Yoshizawa, S., Tsukamoto, T., Sudo, Y.
  • Organizer: 日本生物物理学会中四国支部講演会
• [Presentation] Subunit-subunit interaction in a light-driven proton pump from a thermophilic bacterium: Effects on hydrogen bond of the chromophore (2017)
  • Author(s): Shionoya, T., Mizuno, M., Tsukamoto, T., Sudo, Y., Mizutani, Y.
  • Organizer: The 6th Asian Spectroscopy Conference
  • Int'l Joint Research
• [Presentation] Demonstration of a light-driven SO42- transporter from the cyanobacterium and its spectroscopic characterization (2017)
  • Author(s): Niho, A., Yoshizawa, S., Tsukamoto, T., Kurihara, M., Tahata, S., Nakajima, Y., Mizuno, M., Kuramochi, H., Tahara, T., Mizutani, Y., Sudo, Y.
  • Organizer: International Symposium on Biophysics of Rhodopsins
  • Int'l Joint Research
• [Presentation] Functional and spectroscopic characterization of RmXeR, a xenorhodopsin from a marine bacterium Rubricoccus marinus SG-29T (2017)
  • Author(s): Inoue, S., Yoshizawa, S., Tsukamoto, T., Sudo, Y.
  • Organizer: International Symposium on Biophysics of Rhodopsins
  • Int'l Joint Research
• [Presentation] Cl--pumping photoreaction of a bacterial halide-ion pumping rhodopsin with an archaeal-type TSA motif (2016)
  • Author(s): Tsukamoto, T., Yoshizawa, S., Kikukawa, T., Demura, M., Sudo, Y.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] Functional characterization of a microbial rhodopsin from the marine eubacterium Rubricoccus marinus SG-29 (2016)
  • Author(s): Inoue, S., Yoshizawa, S., Tsukamoto, T., Sudo, Y.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] Identification of thermostabilizing mutations for thermophilic rhodopsin based on statistical thermodynamics (2016)
  • Author(s): Nemoto, S., Yasuda, S., Mizutani, K., Tsukamoto, T., Sudo, Y., Kinoshita, M., Murata, T.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] Structural basis for high thermal stability and efficient optogenetic function of thermophilic rhodopsin (2016)
  • Author(s): Tsukamoto, T., Mizutani, K., Hasegawa, T., Takahashi, M., Honda, N., Hashimoto, N., Shimono, K., Miyauchi, S., Takagi, S., Hayashi, S., Murata, T., Sudo, Y.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] A light-dependent Cl-/H+ antiport activity in anion channelrhodopsin-2 (2016)
  • Author(s): Doi, S., Tsukamoto, T., Chowdhury, S., Yoshizawa, S., Yamanaka, A., Sudo, Y.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] Functional significance of trimerization in Cl- pumping properties of halorhodopsin examined by nanodisc reconstitution (2016)
  • Author(s): Hashimoto, E., Suzuki, K., Yamamoto, A., Tsukamoto, T., Uchida, T., Kikukawa, T., Demura, M., Ishimori, K.
  • Organizer: 日本生物物理学会第54回年会
  • Place of Presentation: つくば市
  • Year and Date: 2016-11-25
• [Presentation] 高度好熱菌Thermus thermophilus 由来サーモフィリックロドプシンの物性研究 (2016)
  • Author(s): 塚本卓, 本田尚也, 西川佳吾, 林重彦, 村田武士, 須藤雄気
  • Organizer: 日本生物物理学会第８回中四国支部講演会
  • Place of Presentation: 高松市
  • Year and Date: 2016-05-28
• [Presentation] 様々な物理化学的刺激に対する安定性の比較から探る微生物型ロドプシンの安定化原理の解明 (2016)
  • Author(s): 金原加苗, 吉澤晋, 本田尚也, 塚本卓, 須藤雄気
  • Organizer: 日本生物物理学会第８回中四国支部講演会
  • Place of Presentation: 高松市
  • Year and Date: 2016-05-28
• [Presentation] アニオンチャネルロドプシン２は光依存性Cl-/H+対向輸送体として機能する (2016)
  • Author(s): 土井聡子, 塚本卓, 吉澤晋, 須藤雄気
  • Organizer: 日本生物物理学会第８回中四国支部講演会
  • Place of Presentation: 高松市
  • Year and Date: 2016-05-28
• [Presentation] 時間分解赤外分光法を用いた熱耐性ロドプシンTRの光反応解析 (2016)
  • Author(s): 黒井邦巧, 塚本卓, 本田尚也, 須藤雄気, 古谷祐詞.
  • Organizer: 日本生物物理学会中部支部講演会
  • Place of Presentation: 岡崎コンファレンスセンター
  • Year and Date: 2016-02-29
• [Presentation] Time-Resolved Infrared Study on the Photoreaction of Thermophilic Rhodopsin (TR) (2015)
  • Author(s): Kuroi, K., Tsukamoto, T., Honda, N., Sudo, Y., Furutani, Y.
  • Organizer: 第9回分子科学討論会
  • Place of Presentation: 東京工業大学 大岡山キャンパス
  • Year and Date: 2015-09-16
• [Presentation] X-ray Crystal Structure of TR: Implications for High Thermal Stability and High-Performance Optogenetic Availability (2015)
  • Author(s): Tsukamoto, T., Mizutani, K., Takahashi, M., Hasegawa, T., Hashimoto, N., Hayashi, S., Takagi, S., Murata, T., Sudo, Y.
  • Organizer: 日本生物物理学会第53回年会
  • Place of Presentation: 金沢大学 角間キャンパス 自然科学本館
  • Year and Date: 2015-09-13
• [Remarks] 研究室HP
  • URL: http://altair.sci.hokudai.ac.jp/infana/
• [Remarks] 岡山大学大学院医歯薬学総合研究科・生体物理化学研究室HP
  • URL: http://www.pharm.okayama-u.ac.jp/lab/bukka/index.html
• [Remarks] researchmap
  • URL: http://researchmap.jp/takashi_tsukamoto/
• [Remarks] ResearcherID
  • URL: http://www.researcherid.com/rid/F-3993-2017
• [Remarks] 北海道大学大学院先端生命科学研究院・生物情報解析科学研究室HP
  • URL: http://altair.sci.hokudai.ac.jp/infana/
• [Remarks] 研究室HP
  • URL: http://www.pharm.okayama-u.ac.jp/lab/bukka/index.html
• [Remarks] 岡山大学薬学部HP
  • URL: http://www.pharm.okayama-u.ac.jp