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

2013 Fiscal Year Final Research Report

• 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
• 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

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 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, S., Tamogami, J., Kikukawa, T., Okamoto, H., Shimono, K., Miyauchi, S., Demura, M., Nara, T., Kamo, N
  • Journal Title: Biophys. Chem Volume: 172 Pages: 61-67
  • DOI: 10.1016/j.bpc.2013.01.001
  • 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 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 Pages: 2906-2915
  • DOI: 10.1016/j.bpj.2012.05.008
  • 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, Y., Kikukawa, T., Tsukamoto, T., Kamiya, M., Aizawa, T., Kawano, K., Miyauchi, S., Kamo, N., Demura, M
  • Journal Title: Biochim. Biophys. Acta Volume: 1808 Pages: 2905-2912
  • DOI: 10.1016/j.bbamem.2011.08.035
  • Peer Reviewed
• [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] 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)
  • Year and Date: 20120930-1005
• [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
• [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
  • Total Pages: 24-33
  • Publisher: エヌ・ティー・エス, オプトジェネティクス