| Project/Area Number |
61430027
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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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 | Kyoto University |
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Principal Investigator |
AKASAKA Kazuyuki Department of Chemistry, Faculty of Science, Kyoto University, 理学部, 助教授 (50025368)
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| Co-Investigator(Kenkyū-buntansha) |
NAITO Akira Department of Chemistry, Faculty of Science, Kyoto University, 理学部, 助手 (80172245)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥24,600,000 (Direct Cost: ¥24,600,000)
Fiscal Year 1987: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1986: ¥19,600,000 (Direct Cost: ¥19,600,000)
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| Keywords | Internal motions in proteins / Stable isotope labeling of proteins / Site-directed mutagenesis / Deuterium NMR / Subtilisin inhibitor / 酵素-阻害剤相互作用 / プロテアーゼインヒビター / 個体タンパク質の水和 / 重水素化タンパク質 / タンパク質 / 内部運動 / トリプトファン / 重水素化 / 放線菌 / 微生物培養 |
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| Research Abstract |
By combination of site-directed mutagenesis and biosynthesis, either one or all of the three methyl groups of the three Met residues per subunit of Streptomyces subtilisin inhibitor (SSI) were selectively replaced with deuterons. By measuring deuterium NMR of the above samples of SSI, detailed molecular motions of the three Met residues were eluucided both in solid state and in solution. When hydration takes place in the solid state, the side chains of Met 70 (P4 site) and 73 (P1 site) in the cr1cial enzyme-binding region undergo large amplitude motions at 1 MHz order. Even when the inhibitor formed a complex with subtilisin BPN', a limited internal motion with a conic angle of about 20 degrees was found to exist within the P1 and P4 pockets of the enzyme. The existence of a motion of SSI in the enzyme-inhibitor complex indicates the "100seness" of fitting between the two molecules, which seems to go along well with the broad specificity of subtilisin BPN'. In addition, the above NMR results has clearyl indicated that such internal motions are brought about only by hydration.
Deuterium NMR of a deuterated single Trp residue of SSI in solution revealed existence of rapid internal motin in the region of Trp in some fraction of SSI. Together with the result on deuterated Met in SSI, this study indicates that deuterium NMR is also very useful to probe internal motions in proteins in solution.
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