| Project/Area Number |
10480159
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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 |
Structural biochemistry
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| Research Institution | Hiroshima University |
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Principal Investigator |
GEKKO Kunihiko Hiroshima Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (10023467)
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| Co-Investigator(Kenkyū-buntansha) |
IWAKURA Masahiro National Institute of Bioscience and Human-Technology, Research Director, 生命工学工業技術研究所, 室長
OHMAE Eiji Hiroshima Univ., Graduate School of Science, Research Associate, 大学院・理学研究科, 助手 (30284152)
KATAYANAGI Katsuo Hiroshima Univ., Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (20291479)
巖倉 正寛 工業技術院, 生命工学工業技術研究所, 室長
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | dihydrofolate reductase / dynamics / loop function / mutagenesis / アミノ酸置換 |
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| Research Abstract |
In order to elucidate the structure-dynamics-function relationships of protein, we investigated the effects of mutation and ligand binding of E.Coli dihydrofolate reduactase (DHFR) on the enzymatic function, adiabatic compressibility, and X-ray structure. Single amino acid substitutions at Gly67, Gly121, and Ala145 in three different flexible loops induced large changes in stability and adiabatic compressibility, indicating the modified flexibility of the structure. The B-factor and cavity distribution at sites far from the mutation sites were largely modified by these mutations, indicating that the local changes in structure due to mutation extend to overall of the protein molecule. The compressibility of the mutants increased with increasing the total cavity volume, indicating the importance of atomic packing in generating the protein dynamics. Although the mutation sites are largely separated from the active site, the mutants having a large compressibility showed high enzymatic activity dominantly due to the enhanced catalytic rate. High pressure NMR experiments revealed that the flexible loops and hinge-motion of active-sites including cofactor-binding domain are strongly influenced by pressure and the equilibrium of two conformers is shifted to open form under high pressure. The compressibility of DHFR changed on ligand binding with the change in the cavity volume, reflecting the characteristic flexibility of the intermediates in the reaction coordinate. These results demonstrate that the local structural changes due to mutation and ligand binding affect the dynamics and function of protein molecule through the modified atomic packing (cavity) and that the flexible loops play important role in protein dynamics and function.
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