Analysis of the elementary processes in the light-driven Cl- pump of halorhodopsin

• Project/Area Number: 23510251
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: Hokkaido University
• Principal Investigator: KIKUKAWA Takashi 北海道大学, 先端生命科学研究科(研究院), 講師 (20281842)
• Co-Investigator(Kenkyū-buntansha): MIYAUCHI Seiji 東邦大学, 薬学部, 教授 (30202352)
• Co-Investigator(Renkei-kenkyūsha): TAMOGAMI Jun 松山大学, 薬学部, 助教 (30580089)
• Project Period (FY): 2011 – 2013
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2011: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: ハロロドプシン / フォトサイクル / アニオンポンプ / 膜蛋白質 / レチナール / イオンポンプ

Research Abstract

Halorhodopsin (HR) is an inward light-driven Cl- pump in the membrane of halophilic archaea. Illumination triggers the photoreaction of HR called photocycle where the intermediates of K, L1, L2, N, O, NpHR' form and decay sequentially. During this cyclic reaction, HR releases Cl- to the cytoplasmic (CP) medium and captures another Cl- from the extracellular medium. To examine the elementary processes for Cl- translocation, we analyzed the photocycle using the flash-photolysis technique and obtained the following results: 1) Cl- release and uptake occur during N to O and O to NpHR' transitions, respectively. The subsequent NpHR' to original NpHR transition involves Cl- displacement inside the protein. 2) The Cl- binding affinity decreases at the Cl- releasing step. This decreased binding affinity contributes to maintain the Cl- pumping activity even in the presence of the interior negative membrane potential.

Research Products

• [Journal Article] Role of Thr218 in the Light-Driven Anion Pump Halorhodopsin from Natronomonas pharaonis (2013)
  • Author(s): Shibasaki, K., Shigemura, H., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Kamo, N., Demura, M
  • Journal Title: Biochemistry Volume: 52 Issue: 51 Pages: 9257-9268
  • DOI: 10.1021/bi401295e
  • Peer Reviewed
• [Journal Article] Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry (2013)
  • Author(s): Hayashi, Saori
  • Journal Title: Biophysical Chemistry Volume: 172 Pages: 61-67
  • DOI: 10.1016/j.bpc.2013.01.001
  • Peer Reviewed
• [Journal Article] Physicochemical Studies of Bacteriorhodopsin Reconstituted in Partially Fluorinated Phosphatidylcholine Bilayers (2013)
  • Author(s): Yoshino, M., Kikukawa, T., Takahashi, H., Takagi, T., Yokoyama, Y., Amii, H., Baba, T., Kanamori, T., Sonoyama, M.
  • Journal Title: J. Phys. Chem. B Volume: 117 Issue: 18 Pages: 5422-5429
  • DOI: 10.1021/jp311665z
  • Peer Reviewed
• [Journal Article] Exporing the active site site structure of a photoreceptor protein by raman optical activity (2013)
  • Author(s): Unno Masashi, kikukawa takashi, Kumauchi Masato, Kamo Naoki
  • Journal Title: Journal of Physical Chemistry. Volume: 117巻 Issue: 5 Pages: 1321-325
  • DOI: 10.1021/jp4001187
  • Peer Reviewed
• [Journal Article] Salt bridge in the conserved His-Asp cluster in Gloeobacter rhodopsin contributes to trimer formation (2013)
  • Author(s): Tsukamoto, T., Kikukawa, T., Kurata, T., Jung, K-H, Kamo, N., Demura, M.
  • Journal Title: FEBS Lett. Volume: 587 Issue: 4 Pages: 322-327
  • DOI: 10.1016/j.febslet.2012.12.022
  • NAID: 120005228320
  • Peer Reviewed
• [Journal Article] Dynamics of Dangling Bonds of Water Molecules in pharaonis Halorhodopsin during Chloride Ion Transportation (2012)
  • Author(s): Furutani, Y., Fujiwara, K., Kimura, T., Kikukawa, T., Demura, M., Kandori, H
  • Journal Title: J. Phys. Chem. Lett Volume: 3 Issue: 20 Pages: 2964-2969
  • DOI: 10.1021/jz301287n
  • Peer Reviewed
• [Journal Article] Homo-trimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System (2012)
  • Author(s): Tsukamoto, T., Sasaki, T., Fujimoto, K. J., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Kamo, N., Demura, M
  • Journal Title: Biophys. J Volume: 102 Issue: 12 Pages: 2906-2915
  • DOI: 10.1016/j.bpj.2012.05.008
  • Peer Reviewed
• [Journal Article] Photoinduced proton release in proteorhodopsin at low pH: The possibility of a decrease in the pKa of Asp227 (2012)
  • Author(s): Tamogami Jun, Kikukawa Takashi, NaraToshifumi , Shimono Kazumi, Demura Makoto, Kamo Naoki
  • Journal Title: Biochemistry Volume: 51巻 Issue: 46 Pages: 9290-9301
  • DOI: 10.1021/bi300940p
  • Peer Reviewed
• [Journal Article] "The influence of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I" (2012)
  • Author(s): L. Reissig, T. Iwata, T. Kikukawa, M. Demura, N. Kamo, H. Kandori and Y. Sudo
  • Journal Title: Biochemistry Volume: 51 Issue: 44 Pages: 8802-8813
  • DOI: 10.1021/bi3009592
  • Peer Reviewed
• [Journal Article] Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacteium salinarum by hydroxylamine (2012)
  • Author(s): Tamogami Jun
  • Journal Title: Journal of Photochemistry and Photobiology B Volume: 106 Pages: 87-94
  • DOI: 10.1016/j.jphotobiol.2011.10.009
  • Peer Reviewed
• [Journal Article] Expression of salinarum halorhodopsin in Escherichia coli cells : Solubilization in the presence of retinal yields the natural state (2011)
  • Author(s): Yamashita Yasutaka
  • Journal Title: Biochimica et Biophysica Acta-Biomembranes Volume: 1808 Issue: 12 Pages: 2905-2912
  • DOI: 10.1016/j.bbamem.2011.08.035
  • Peer Reviewed
• [Journal Article] Photochemistry of acetabularia rhodopsin II from a marine plant, Aceta bularia acetabulum (2011)
  • Author(s): Kikukawa Takashi
  • Journal Title: Biochemistry Volume: 50 Issue: 41 Pages: 8888-8898
  • DOI: 10.1021/bi2009932
  • Peer Reviewed
• [Journal Article] Crystal structure of the Eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II from Marine Algae (2011)
  • Author(s): Wada Takashi
  • Journal Title: Journal of Molecular Biology Volume: 411 Issue: 5 Pages: 986-998
  • DOI: 10.1016/j.jmb.2011.06.028
  • Peer Reviewed
• [Presentation] 二量体化した Di-O-Tetradecylphosphatidylcholine への膜タンパ ク質バクテリオロドプシンの再構成 (2014)
  • Author(s): 土田直之、高木俊之、菊川峰志、高橋 浩、金森敏幸、園山正史
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] Analysis of Cl-release and uptake steps of light-driven Cl-pump Natronomonas pharaonis halorhodopsin (2013)
  • Author(s): Kikukawa, T
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館(京都市)
  • Year and Date: 2013-10-29
• [Presentation] Role of Thr218 in light-driven Cl- pump mechanism of Natronomonas pharaonis halorhodopsin (2013)
  • Author(s): Shibasaki, K
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館(京都市)
  • Year and Date: 2013-10-29
• [Presentation] 部分フッ素化リン脂質・非フッ素化リン脂質二成分混合膜に再構成したバクテリオロドプシンの脂質選択性 (2013)
  • Author(s): 吉野 賢、菊川 峰志、高橋 浩、 高木 俊之、網井 秀樹、金森 敏幸、 園山 正史
  • Organizer: 第13回に本蛋白質科学会年会
  • Place of Presentation: とりぎん文化会館（鳥取市）
• [Presentation] 光駆動型 Cl- ポンプハロロドプシンの光反応サイクルにおける荷電性残基の役割 (2013)
  • Author(s): 大塚 瞭、菊川 峰志、神谷 昌克、 相沢 智康、河野 敬一、加茂 直樹、 出村 誠
  • Organizer: 第13回に本蛋白質科学会年会
  • Place of Presentation: とりぎん文化会館（鳥取市）
• [Presentation] ハロロドプシンの光反応サイクルに現れる膜電位依存性 (2013)
  • Author(s): 日下部 力、菊川 峰志、神谷 昌克、 相沢 智康、河野 敬一、井原 邦夫、 加茂 直樹、出村 誠
  • Organizer: 第13回に本蛋白質科学会年会
  • Place of Presentation: とりぎん文化会館（鳥取市）
• [Presentation] Photochemical reaction in Acetabularia rhodopsin I (2013)
  • Author(s): Tamogami, J., Kikukawa, T., Shimono, K., Kimura-Someya, T., Shirouzu, M., Yokoyama, S., Kamo, N.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都市）
• [Presentation] Analysis of Cl- release and uptake steps of light-driven Cl- pump Natronomonas pharaonis halorhodopsin (2013)
  • Author(s): Kikukawa, T., Kusakabe, C., Kokubo, A., Tsukamoto, T., Kamiya, M., Aizawa, T., Ihara, K., Kamo, N., Demura, M.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都市）
• [Presentation] Role of Thr218 in light-driven Cl- pump mechanism of Natronomonas pharaonis halorhodopsin (2013)
  • Author(s): Shibasaki, K., Shigemura, H., Kikukawa, T., Kamiya, M., Aizawa, T., Kamo, N., Demura, M.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都市）
• [Presentation] A Biophysical Study of Bacteriorhodopsin in Pseudocyclic Phosphatidylcholine Liposome (2013)
  • Author(s): Sonoyama, M., Yoshioka, S., Tsuchida, N., Takagi, T., Takahashi, H., Kikukawa, T., Kanamori, T.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都市）
• [Presentation] Low affinity of bacteriorhodopsin to partially fluorinated phosphatidylcholine in reconstituted membrane (2013)
  • Author(s): Yoshino, M., Kanayama, K., Kikukawa, T., Takagi, T., Takahashi, H., Yokoyama, Y., Amii, H., Kanamori, T., Somoyama, M.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都市）
• [Presentation] リン脂質リポソームに再構成したバクテリオロドプシンの構造・機能に対するアシル鎖部分フッ素化の影響 (2013)
  • Author(s): 吉野賢、菊川峰志、高橋浩、高木俊之、横山泰範、網井秀樹、金森敏之、園山正史
  • Organizer: 第64回コロイドおよび界面化学討論会
  • Place of Presentation: 名古屋工業大学（名古屋市）
• [Presentation] Native-like reconstitution of bacteriorhodopsin into novel partially fluorinated phosphatidylcholine lipid bilayer (2013)
  • Author(s): Sonoyama, M., Yoshino, M., Kikukawa, T., Takahashi, H., Takagi, T., Yokoyama, Y., Amii, H., Kanamori, T.
  • Organizer: The International Conference on Structural Genomics 2013
  • Place of Presentation: 京王プラザホテル（札幌市）
• [Presentation] 部分フッ素化リン脂質を含む二成分系リン脂質混合膜における 膜タンパク質バクテリオロドプシンの分配挙動 (2013)
  • Author(s): 吉野賢、金山賢治、高橋浩、高木俊之、菊川峰志、横山泰範、網井秀樹、金森敏之、園山正史
  • Organizer: 第40回生体分子科学討論会
  • Place of Presentation: 大阪大学（吹田市）
• [Presentation] When does halorhodopsin capture and release Cl-during photocycle? Examination by spectrum changes of an intrinsic voltage-sensitive dye, bacterioruberin (2012)
  • Author(s): Kikukawa, T
  • Organizer: 15th International Conference on Retinal Proteins
  • Place of Presentation: Monte Verita Congress Center(Swizarland)
• [Presentation] When does halorhodopsin capture and release Cl- during photocycle? Examination by spectrum changes of an intrinsic voltage-sensitive dye, bacterioruberin (2012)
  • Author(s): Kikukawa, T., Kokubo, A., Tsukamoto, T., Ihara, K., Kamo, N., Demura, N.
  • Organizer: 15th International Conference on Retinal Proteins
  • Place of Presentation: Monte Verita Congress Center(Swizarland)
• [Presentation] The effect of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I (2012)
  • Author(s): Reissig, L., Iwata, T., Kikukawa, T., Demura, M., Kamo, N., Kandori, H., Sudo, Y.
  • Organizer: 15th International Conference on Retinal Proteins
  • Place of Presentation: Monte Verita Congress Center(Swizarland)
• [Presentation] The Proton Release in Proteorhodopsin at Low pH: Decrease in pKa of Asp227 in the excited state (2012)
  • Author(s): Tamogami, J., Kikukawa, T., Nara, T., Shimono, K., Demura, M., Kamo, N.
  • Organizer: 15th International Conference on Retinal Proteins
  • Place of Presentation: Monte Verita Congress Center(Swizarland)
• [Presentation] Homo-trimeric structure of Gloeobacter rhodopsin is regulated by the protonation state of Asp121, a counterion of the protonated Schiff base (2012)
  • Author(s): Tsukamoto, T., Kikukawa, T., Kurata, T., Jung, K-H., Kamo, N., Demura, M.
  • Organizer: 15th International Conference on Retinal Proteins
  • Place of Presentation: Monte Verita Congress Center(Swizarland)
• [Presentation] Natronomonas pharaonis 由来のセンサリーロドプシンII の光誘起プロトン移動における塩化物イオンの役割 (2012)
  • Author(s): Tamogami, J., Iwano, K., Matsuyama, A., Kikukawa, T., Demura, M., Shimono, K., Nara, T., Kamo, N.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] アナべナセンサリーロドプシンの光誘起プロトン移動反応の解析 (2012)
  • Author(s): Hasemi, T., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Jung, K-H., Kamo, N., Demura, M.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] グロエオバクターロドプシンの三量体形成に関する研究 (2012)
  • Author(s): Tsukamoto, T., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Jung, K-H., Kamo, N., Demura, M.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] 時間分解赤外分光法によるファラオニスハロロドプシンの機能発現過程での水の水素結合ダイナミクス (2012)
  • Author(s): Fujiwara, K., Kimura, T., Kikukawa, T., Demura, M., Kandori, H., Furutani, Y.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] 部分フッ素化ホスファチジルコリン小胞に再構成されたバクテリオロドプシンの液晶相における分子集合体 (2012)
  • Author(s): Yoshino, M., Kanayama, K., Kikukawa, T., Takagi, T., Takahashi, H., Baba, T., Kanamori, T., Sonoyama, M.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] Laser flash photolysis study on the decay kinetics of the Mphotointermediate of bacteriorhodopsin in diheptanoylphosphocholine micelles (2012)
  • Author(s): Sonoyama, M., Kuwabara, Y., Kikukawa, T.
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] 三次元固体NMR 法による膜タンパク質ハロロドプシンの研究 (2012)
  • Author(s): 田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場（名古屋市）
• [Presentation] 短鎖リン脂質ジヘプタノイルホスファチジルコリンを用いた可溶化による膜タンパク質バクテリオロドプシンAsp96 のpKa の変化 (2012)
  • Author(s): 園山正史, 桑原由美子, 菊川峰志
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場（名古屋市）
• [Presentation] ハロロドプシンの光反応サイクル中に起こるカロテノイドの吸光度変化 (2012)
  • Author(s): 菊川峰志,小久保麻実, 塚本卓, 井原邦夫, 加茂直樹, 出村誠
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場（名古屋市）
• [Presentation] APPLICATION OF NOVEL PARTIALLY FLUORINATED PHOSPHOLIPID: RECONSTITUTION OF MEMBRANE PROTEIN BACTERIORHODOPSIN INTO LIPOSOME (2012)
  • Author(s): Sonoyama, M., Yoshino, M., Takagi, T., Kikukawa, T., Takahashi, H., Baba, T., Kanamori, T.
  • Organizer: 20th International symposium on fluorine chemistry
  • Place of Presentation: 京都テルサ（京都市）
• [Presentation] Volume change during the Cl- transport of Natronomonas pharaonis halorhodopsin (2011)
  • Author(s): Shibasaki, K
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学(姫路市)
  • Year and Date: 2011-09-16
• [Presentation] 海洋細菌由来のロドプシン様タンパク質プロテオロドプシンの低pH条件下における速いプロトン放出機構
  • Author(s): 田母神淳，菊川峰志，下野和実，奈良敏文，宗行英朗，加茂直樹
  • Organizer: 日本薬学会第132年会
  • Place of Presentation: 北海道大学（札幌市）
• [Presentation] 非線形サンプリングを用いた膜タンパク質の固体NMR測定
  • Author(s): 田巻初，江川文子，松木陽，神谷昌克，菊川峰志，相沢智康，河野敬一，藤原敏道，出村誠
  • Organizer: 第11回蛋白質科学会年会
  • Place of Presentation: ホテル阪急エキスポパーク（大阪府
• [Presentation] キサントロドプシンの光誘起プロトン移動の解析
  • Author(s): 菊川峰志，佐藤由美，田母神淳，下野和実，神谷昌克，相沢智康，河野敬一，出村誠，加茂直樹
  • Organizer: 第11回蛋白質科学会年会
  • Place of Presentation: ホテル阪急エキスポパーク（大阪府）
• [Presentation] Volume change during the Cl- transport of Natronomonas pharaonis halorhodopsin
  • Author(s): 柴崎宏介，菊川峰志，神谷昌克，相沢智康，河野敬一，加茂直樹，出村誠
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学（姫路市）
• [Presentation] The fast proton release of proteorhodopsin at low pH
  • Author(s): 田母神淳，菊川峰志，下野和実，奈良敏文，宗行英朗，加茂直樹
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学（姫路市）
• [Presentation] Role of Ser171 for the stabilization of NpHR trimer
  • Author(s): 塚本卓，寺田浩太，中川摩里恵，田巻初，神谷昌克，菊川峰志，相沢智康，河野敬一，出村 誠
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学（姫路市）
• [Presentation] Reconstitution of bacteriorhodopsin into partially fluorinated phosphatidylcholine liposomes
  • Author(s): 吉野賢，菊川峰志，横山泰範，高木俊之，高橋浩，出村誠，馬場照彦，金森敏幸，園山正史
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学（姫路市）
• [Presentation] Decay kinetics of the M photointermediate of bacteriorhodopsin in short chain phosphatidylcholine micelles
  • Author(s): 桑原由美子，福元美奈，菊川峰志，出村誠，園山正史
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学（姫路市）
• [Presentation] 短鎖リン脂質ジヘプタノイルホスファチジルコリンミセル中の膜タンパク質バクテリオロドプシンの不均一性
  • Author(s): 桑原由美子，菊川峰志，出村誠，園山正史
  • Organizer: 日本化学会第92春季年会
  • Place of Presentation: 慶應義塾大学（横浜市）
• [Presentation] STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN RECONSTITUTED INTO PARTIALLY FLUORINATED PHOSPHATIDYLCHOLINE LIPOSOME
  • Author(s): Yoshino, M, Kikukawa, T., Takagi, T., Yokoyama, Y., Takahashi, H., Baba, T., Kanamori, T., Demura, M., Sonoyama, M.
  • Organizer: 「揺らぎと生体機能」第５回公開シンポジウム
  • Place of Presentation: 東大寺総合文化センター（奈良市）
• [Book] -光工学と遺伝学による行動制御技術の最前線- (2013)
  • Author(s): 出村誠, 菊川峰志, 加茂直樹
  • Publisher: エヌ・ティー・エス, オプトジェネティクス
• [Book] オプトジェネティクス－光工学と遺伝学による行動制御技術の最前線－ (2013)
  • Author(s): 出村誠, 菊川峰志, 加茂直樹
  • Publisher: エヌ・ティー・エス
• [Book] Photo-induced Proton Transfers of Microbial Rhodopsins, In Molecular Photochemistry - Various Aspects (Dr. Satyen Saha, Ed.) (2012)
  • Author(s): Kikukawa, T., Tamogami, J., Shimono, K., Demura, M., Nara, T., Kamo, N.
  • Publisher: InTech