2001 Fiscal Year Final Research Report Summary
Elucidation of the Cl-transport mechanism of halorthodopsin by time-resolved electric and spectroscopic measurements
| Project/Area Number |
11680653
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Tokyo Institute of Technology |
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Principal Investigator |
MUNEYUKI Eiro Chemical Resources Laboratory, Tokyo Institute of Technology, Research Associate, 資源化学研究所, 助手 (80219865)
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| Project Period (FY) |
1999 – 2001
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| Keywords | halorhodopsin / ion pump / photocych / photovoltage / spectroscopic measurements |
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| Research Abstract |
First of all, we improved the photovoltage measurements system so that it enabled us to examine the photovoltage generation from 1 μsec to 500 msec.
Then, using the time-resolved photovoltage measurement system, we examined photovoltage kinetics of halorthodopsin from Halobacterium salinarum. As a function of chloride concentrations. The photovoltage consisted of two major components ; one with a sub-ms range time constant and the other with ms range time constant with different amplitudes., These components exhibited different Cl^- concentration dependencies (0.1 M to 9 M). We found that the time constant for the fast component was relatively independent of the Cl^- concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl^- concentrations. The fast and the slow processes were attributed to charge (Cl^-) movements within the protein and related to Cl^- ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that
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they correspond to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl^- concentration dependence and the Cl^- concentration dependence of its time constant suggested a weak and highly cooperative Cl^- binding site(s) at cytoplasmic side (apparent K_D of about 5 M and Hill coefficient ≧ 5). The Cl^- concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be > 100 ms.
Electrogenic activity of the halorhodopsin (hR) from shark strain was also examined. After absorbing hR-overproduced membrane fraction onto a thin polymer film, we measured photoelectric current upon illumination at various Cl^- concentrations. The amplitude of the photoelectric current exhibited a bell-shaped Cl^- concentration dependency. It was found that R108Q mutant also exhibited photoelectric current at a high Cl^- concentration. The result indicates that R108 is important for uptaking Cl^- from the medium, but once Cl^- is located near the Schiff base, hR can exhibit electrogenicity even without R108. Less
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[Publications] Mitome, N, Ono, S., Suzuki, T., Shimabukuro, K., Muneyuki, E, Yoshida, M.: "Presence of phosphate at a catalytic site suppresses the formation of the MgADP inhibited form of F_1-ATPase"Eur. J. Biochem.. 269. 53-60 (2002)
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「研究成果報告書概要(和文)」より
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[Publications] Hirono-Hara, Y., Noji, H., Nishiura, M., Muneyuki, E., Hara, K-Y., Yasuda, R., Kinosita, K., Jr., Yoshida, M.: "Pause and rotation of F_1-ATPase during catalysis"Proc. Natl Acad. Sci. USA. 98. 13649-13654 (2001)
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[Publications] Masaike, T., Noji, H., Muneyuki, E., Yassuda, R., Kinosita Jr.K., Yoshida, M.: "Rotation of F_1-ATPase and the hinge residues of the β subunit"J. Exper.Biol.. 203. 1-8 (2000)
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[Publications] Bald, D., Muneyuki, E., Amano, T., Kruip, J., Hisabori, T., Yoshida, M.: "The noncatalytic site-deficient α_3β_3γ subcomplex and FoF_1-ATP synthase can continuously catalyze ATP hydrolysis when Pi is present"Eur. J. Biochem.. 262. 563-568 (1999)
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[Publications] Amano, T., Matsui, T., Muneyuki, E., Noji, H., Yoshida, M., Hisabori, T.: "α_3β_3γ Complex of F_1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites"Biochem. J.. 343. 135-138 (1999)
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