| Project/Area Number |
12480197
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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 | The University of Tokyo |
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Principal Investigator |
KUWAJIMA Kunihiro Graduate School of Science, Department of Physics, Professor, 大学院・理学系研究科, 教授 (70091444)
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| Co-Investigator(Kenkyū-buntansha) |
新井 宗仁 東京大学, 大学院・理学系研究科, 助手 (90302801)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2002: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2000: ¥9,400,000 (Direct Cost: ¥9,400,000)
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| Keywords | Folding / Temperature jump / Molecular Dynamics / Staphylococcal nuclease / α-Lactalbumin / Ca^<2+>-binding lysozyme / Unfolding / Denaturation / 蛋白質フォールディング / 分子動力学シミュレーション / 高圧 / スタフィロコッカルヌクレアーゼ / リゾチーム |
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| Research Abstract |
To elucidate the folding mechanism of proteins, the present study has been carried out with two objectives. (1) For the purpose of monitoring refolding kinetics of globular proteins in a time regime of microsecond to millisecond, we have developed a high-pressure Joule heating temperature-jump apparatus, tested the apparatus and improved its capability. (2) For the purpose of describing the folding process of proteins at an atomic level, we have carried out unfolding simulations of a protein at a high temperature (400〜600 K), and compared the results with known experimental data.
The following results were obtained.
(1) The high-pressure temperature-jump apparatus developed in the present study allows us to monitor the reactions by both ultraviolet absorption and fluorescence spectroscopy, and the pressure achieved was 1,800 atm at 25℃ and 1,200 atm at -4℃. Because many proteins are in the cold denatured state at -4℃ and 1,200 atm, it will be possible to monitor the folding reaction of the proteins in a microsecond time regime by the temperature-jump method.
(2) We have studied the refolding reaction of proline-free pseudo wild-type staphylococcal nuclease, which will be used as a model protein in the temperature-jump measurements. As a result, this protein has been found to refold along multiple parallel reaction pathways from the denatured state although the protein does not have proline residues. This finding is the first case in which the presence of multiple parallel pathways in protein folding is clearly shown.
(3) It has been known experimentally that a recombinant form of goat α-lactalbumin unfolds 100-fold faster than its authentic form. Here, we have reproduced the experimental results by unfolding simulations by molecular dynamics. Because of the presence of an additional methione residue at the N terminus in the recombinant protein, the structure near the N-terminus has been found to show significantly large fluctuations even at room temperature.
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