Rotation generation and control of the direction in ATR synthase
| Project/Area Number |
18570139
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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 |
Functional biochemistry
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| Research Institution | Nagahama Institute of Bio-Science and Technology |
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Principal Investigator |
KIHARA Atsuko Nagahama Institute of Bio-Science and Technology, Department of Bioscience, Associate Professor (70252715)
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| Co-Investigator(Kenkyū-buntansha) |
NAMBU Takayuki Nagahama Institute of Bio-science and Technology, Department of Bioscience, Assistant Professor (80367903)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | molecular motor / nano-machine / bioenergetics / energy counling / ATP合成酵素 |
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| Research Abstract |
ATP synthase is a nano-size motor enzyme that exchanges proton motive force to ATP synthesis when protons are translocated through the proton pathway, the rotor and stator of the enzyme rotate each other. It is believed that the continuous unidirectional rotation couples with the rotation and the ion transport. The gamma subunit which located at the centre of catalytic sites was possible to regulate the enzyme rotation
The gamma subunit gene of Escherichia call was subjected to region-specific random mutagenesis by using PCR, and they were substituted with the same region of the expression plasmid for the wild-type ATP synthase. We isolated mutants that were not grown on succinate by oxidative phosphorylation. One mutant carrying Ser12Gly replacement was found that ATPase activity was 50% of wild-type; however proton translocation was reduced less than 20%. According to tertiary structure of Fl in the transition state from bovine mitochondria, the Ser-12 residue formed hydrogen bond to the Asp-372 in beta subunit. Interaction between gamma Ser-12 and beta Asp-372 may crucial in regulation of rotation direction and energy coupling. Another mutant having Leu-219 to Pro substitution possibly broke the alpha-helix structure in the carboxyl terminal region of the gamma subunit. The mutant enzyme showed reduced proton transport to compare with its activity of ATP hydrolysis.
Mechanism of the enzyme rotation, rotation generation and stopping, was not known yet. We observed rotations of F1's that indicated decreasing rotation rate previously and from its revertant. While the mutant F1 of Ser-174 to Phe in beta subunit often stopped at the product releasing step, the F1 with Phe-174/Ile-163 did not. Structural calculation of the part around catalytic site in beta subunit revealed Phe residue at the position 174 and Ile-163 strongly interact, by which conformational change triggered by product release in ATP hydrolysis was inhibited.
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Report
(3 results)
Research Products
(18 results)
