1993 Fiscal Year Final Research Report Summary
Analysis of the dynamic conformation change in the light-driven proton pump cross-bridged by S-S bond.
| Project/Area Number |
04680268
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Nagoya University |
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Principal Investigator |
SUGIYAMA Yasuo Department of Biology, School of Science, Nagoya University, Lecturer, 理学部, 講師 (70154507)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Halophilic bacteria / Light-driven proton pump / Retinal protein / Archaerhodopsin / Chimeric archaerhodopsin / Mutated archaerhodopsin / Genetic engineering |
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| Research Abstract |
We are studying the effects of the S-S bond introduced into the loop region connecting the transmembrane helices on the stability and the activity of the light-driven proton pump in Halobacteria to detect the structure change essential for the proton pumping. In this work we expressed the chimeric and the mutated archaeopsin (aO) in E.coli, and the Cys-derivative of bacteriorhodopsin (bR) in Halobacterium salinarium.
1.The hybrid proteins, which were constructed by exchanging the helice (s) of aO-1 with the corresponding one (s) of aO-2, could be refolded into the intact structure and regenerated to the chromophore in vitro. In contrast, the insertion or deletion of 5 amino acids in the loop EF decreased the rate and yield of chromophore regeneration. Furthermore these mutant aRs showed the atypical photocycle. These indicate that the mutagenesis in the loop region may have a defect in the correct packing of the seven transmembrane helices.
2.The bR derivative, L66C/V199C,whose amino acids located at the loop regions connecting helices B and C,and helices F and G were changed to Cys, were produced as purple membrane in H.salinalium and showed the normal photocycle as the wild type bR.Then we examined whether this mutant bR had the S-S bond or not and found that the Cys residue introduced at 199 position was not exposed to the surface region of the protein and, therefore, the S-S bond was not formed. This shows that it is necessary to reform the structure model of bR,proposed by R.Henderson in 1990, at least in the loop FG.
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[Publications] Lukashev, E.P., Govindjee, R., Kono, M., Ebrey, T.G., Sugiyama, Y.and Mukohata, Y.: "pH dependence of the absorption spectra and photochemical transformations of the archaerhodopsins." Photochem.Photobiol.60. 69-75 (1994)
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