| Project/Area Number |
12480201
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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 | Hiroshima University (2001)
Kobe University (2000) |
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Principal Investigator |
GEKKO Kunihiko (2001) Hiroshima Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (10023467)
赤坂 一之 (2000) 神戸大学, 大学院・自然科学研究科, 教授 (50025368)
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| Co-Investigator(Kenkyū-buntansha) |
OHMAE Eiji Hiroshima Univ., Graduate School of Science, Assistant, 大学院・理学研究科, 助手 (30284152)
GO Michiko Nagoya Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (70037290)
月向 邦彦 広島大学, 大学院・理学研究科, 教授 (10023467)
田村 厚夫 神戸大学, 大学院・自然科学研究科, 講師 (90273797)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2001: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2000: ¥8,200,000 (Direct Cost: ¥8,200,000)
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| Keywords | dihydrofolate reductase / compressibility / high pressure NMR / cavity / flexibility / enzyme function / "open"構造 / 原子間の空隙 / NMR化学シフト / モジュール / 蛋白質立体構造 |
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| Research Abstract |
Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate with the aid of coenzyme NADPH in the kinetic reactions cycling through five intermediates. The adiabatic compressibility of DHFR-changes alternatively by binding or releasing these ligands, parallel to the changes in cavity volume, indicating that the cavity plays dominant role in protein dynamics and enzyme function. Single amino acid substitutions at Gly67, Gly121, and Ala145 in three different flexible loops of DHFR induce large changes in compressibility. Compressibility increases with increasing enzyme activity, indicating that protein dynamics positively contributes to the function. The B-factor and cavities at positions far from the mutation sites are influenced and the mutants with a large amount of cavity show large compressibility. High-pressure NMR reveals that pressure induces a local melting of specific structural elements near hydrophobic cavities of the Ras binding domain of RalGDS while the overall folded structure is maintained. A cavity-filling mutation (V103L) of the c-Myb R2 subdomain induces a dramatic increase (in press)ure stability, giving a clear example of the effect of a cavity on dynamics and stability of the protein. Two folded conformers of ubiqutin were first revealed by high pressure-NMR, indicating this technique is very sensitive to the structural change with a small energy difference. These results demonstrate that the characterization of interatomic cavities (pressure effects) gives new insight into protein dynamics and function.
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