| Project/Area Number |
01580258
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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 | Hokkaido University |
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Principal Investigator |
KUWAJIMA Kunihiro Hokkaido University, Department of Polymer Science, Instructor, 理学部, 助手 (70091444)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Protein Folding / Transition State / Staphylococcal Nuclease / Stopped-Flow CD / Site-Directed Mutation / Kinetics / 折りたたみ機構 / スタフィロコッカルヌクレア-ゼA / CDスペクトル / プラスミドベクタ- / 組換えDNA / 部位特異的アミノ酸置換 |
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| Research Abstract |
Investigations of the transition state of folding have primary importance in elucidating the mechanism of protein folding. Here, we have used staphylococcal nuclease (SNase) as a model protein for analysis of the critical structure in the transition state and established an effective expression system to utilize site-directed amino acid replacements for such an analysis. The following results were obtained. (1) Unfolding and refolding of wild-type SNase induced by concentration jumps of urea have been studied by stopped-flow CD measurements in the peptide region. An intermediate that has an appreciable amount of peptide secondary structure accumulates at an early stage of the refolding, but it has also been shown to be significantly less stable than similar intermediates previously observed in other proteins. (2) Effects of specific ligands (Ca^<2+> and pdTp) for SNase on the kinetic folding have been analyzed quantitatively to obtain an insight into the structure of the transition state. Comparison of the present results with those on the two other Ca^<2+>-binding proteins (alpha-lactalbumin and parvalbumin) in previous studies has led to the conclusion that the critical structure in the transition state reflects the hierarchical nature of protein structure folding. (3) An expression-secretion plasmid for SNase in E. Coli, which is useful for making SNase mutants, was constructed by use of a lac-tac tandem promoter followed by the ribosome-binding site and the signal sequence of E. Coli protease III. (4) A mutant SNase (P117G) in which Pro117 was replaced by glycine was made, and its kinetic unfolding and refolding were investigated to study the effect of proline cis-trans isomerism on the kinetics. The proline to glycine mutation simplifies the kinetics of both unfolding and refolding and has been shown to be useful for analysis of the transition state of folding.
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