2004 Fiscal Year Final Research Report Summary
Molecular mechanism of chaperonin-assisted protein folding studied by single molecule imaging.
| 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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| Keywords | molecular chaperon / single molecule imaging / GroEL |
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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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[Journal Article] High-performance liquid chromatographic assay of N^G-monomethly-L-arginine, N^G, N^G-dimethyl-L-arginine, and N^G, N-<G'>-dimethyl-L-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent2005
Author(s)
Nonaka, S., M.Tsunoda, K.Imai, T.Funatsu
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Journal Title
J.Chromatogr. A 1066
Pages: 41-45
Description
「研究成果報告書概要(和文)」より
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[Journal Article] High-performance liquid chromatographic assay of N^G-monomethyl-L-arginine, N^G,N^G-dimethyl-L-arginine, and N^G,N^G-dimethyl-L-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent2005
Author(s)
Nonaka, S., M.Tsunoda, K.Imai, T.Funatsu.
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Journal Title
J Chromatogr.A. 1066
Pages: 41-45
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Specific flow control systems using IR laser indeced sol-gel tranfer of hydrogel2004
Author(s)
Tanaka, J.-I., Y.Shirasaki, M.Tatsuoka, T.Funatsu, S.Watanabe, S.Shoji, T.Edura, J.Mizuno, K.Tsutsui, Y.Wada
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Journal Title
NSTI Nanotechnology Conference and Trade Show-NSTI Nanotech 2004 1
Pages: 296-299
Description
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