| Project/Area Number |
10044092
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
SAITO Hazime Himeji Institute of Technology, Professor, 理学部, 教授 (30100150)
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| Co-Investigator(Kenkyū-buntansha) |
KATAOKA Mikio Nara Institute of Advanced Science and Technology, Professor, 大学, 教授 (30150254)
TUZI Satoru Himeji Institute of Technology, Research Associate, 理学部, 助手 (60227387)
NAITO Akira Himeji Institute of Technology, Associate Professor, 理学部, 助教授 (80172245)
KANDORI Hideki Kyoto University, Assistant Professor, 大学院・理学研究科, 講師 (70202033)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1998: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Membrane proteins / Bacteriorhodopsin / Solid State NMR / FTIR / X-ray diffraction / Photoisomerization / Proton pump |
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| Research Abstract |
Mechanistic studies on proton pump activity of bacteriorhodopsin (bR) were extensively performed under the intensive US-Japan international collaborative program utilizing high-resolution solid-state NMR, infrared spectroscopy, X-ray diffraction and a variety of site-directed mutants.
(1) Solid state NMR : The interhelical loop regions located at the membrane surface undergo conformational fluctuations with correlation times of 10ィイD1-4ィエD1s, because their ィイD113ィエD1C NMR signals of [2-ィイD113ィエD1C]Ala-bR were completely suppressed as a result of interference with magic angle spinning. In addition, major conformational changes were induced in the helix B and B-C loop in these mutants in which M-like state was formed without photo-illumination as a result of protonation to Asp 85. This finding was obtained on the basis of ィイD113ィエD1C NMR spectra of [3-ィイD113ィエD1C]Ala- and [1-ィイD113ィエD1C]Val-labeled bR and D85N and D85N/D96N mutants at higher alkaline pH, because resulting conformational changes were well characterized by means of conformation-dependent displacements of ィイD113ィエD1C NMR Peaks.
(2) Infrared spectroscopy : Structural change of bR accompanied with all-trans to 13-cis photoisomerization were examined by polarized Fourier transform infrared Spectroscopy (FTIR). The measurements of 〔3-ィイD118ィエD1O〕Thr-labeled bR revealed that one of the DィイD22ィエD2O-sensitive bands exhibited ィイD118ィエD1O-induced isotope shift. This means that shortening of hydrogen-bond between the OH group of Thr 89 and carboxyl oxygen atom of Asp 85 was caused by the photoisomerization.
(3) X-ray diffraction : Time-resolved small angle X-ray diffraction studies of bR were extensively analyzed to characterize the deprotonation process of Schiff base after photo illumination. There exist the two forms : rapid and slow components were then ascribed to the presence of the M state and N state, respectively. Further, high-resolution 3D structures of bR are now available for the ground and M-state.
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