| Project/Area Number |
14380320
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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 (2004)
Waseda University (2002-2003) |
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Principal Investigator |
FUNATSU Takashi The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (00190124)
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| Co-Investigator(Kenkyū-buntansha) |
TAGUCHI Hideki The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院・新領域創成科学研究科, 助教授 (40272710)
TADAKUMA Hisashi The University of Tokyo, Graduate School of Engineering, Assistant Professor, 大学院・工学系研究科, 助手 (10339707)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥16,900,000 (Direct Cost: ¥16,900,000)
Fiscal Year 2004: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2003: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2002: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Keywords | molecular chaperon / single molecule imaging / GroEL / シャペロニン / 1分子イメージング / タンパク質間相互作用 |
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| Research Abstract |
Chaperonin GroEL mediates substrate protein folding in a repeated cycle of ATP-driven GroEL-GroES association-dissociation processes. We have succeeded in detecting the processes at the level of a single molecule using total internal reflection fluorescence microscopy (TIRFM). Our results indicated that this process consists of two timers approximately 3 sec and 5 sec of the rate-limiting steps. In order to gain understating of this two timer process, acid-denatured GFP was used as a substrate protein for GroEL and its folding was observed. The result from this observation demonstrated that the substrate folding was inhibited in the first 3 sec period, and revealed a cis ADP*-complex, that is a new complex existed in the 5 sec rate-limiting step.
Furthermore, the existence of a so called football-shaped intermediate complex, consisting of a GroEL bound both sides with GroES, was examined using a new single molecule fluorescence imaging method. In this imaging, nano-holes with 100 nm diameter were fabricated in a metal film deposited on a slide-glass. This was used to localize evanescent field, reducing the fluorescence background, enabling us to image a single fluorescent molecule in the presence of a high concentration of fluorescence molecules. Using this method, processes of Gro-GroES association-dissociation was revealed at the level of a single molecule in the presence of fluorescently labeled 1 μM of GroES.
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