| Project/Area Number |
10480181
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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 | Osaka University |
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Principal Investigator |
GOTO Yuji Osaka University, Institute for Protein Research, Professor, たんぱく質研究所, 教授 (40153770)
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| Co-Investigator(Kenkyū-buntansha) |
YANAGIDA Toshio Osaka University, Faculty of Medicine, Professor, 医学部, 教授 (30089883)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥11,100,000 (Direct Cost: ¥11,100,000)
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| Keywords | Protein / Protein Folding / Single Molecule Analysis / β-Lactoglobulin / β2-Microglobulin / Fluorescence / NMR / Amyloid Fibril / 蛋白質 |
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| Research Abstract |
Single molecular analysis is becoming important as a critical approach for characterizing the structure and function of protein molecules. The aim of this project is to improve our understanding of the mechanism of protein folding and stability by using novel approaches of single molecular analysis.
1. First, to understand the mechanism of GroEL-assisted protein folding, we studied the interaction of fluorescence-labeled GroEL and substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. We, for the first time, demonstrated the direct interaction between GroEL and substrate proteins, which was dissociated upon addition of ATP.
2. To understand the mechanism of amyloid fibril formation, we expressed human β2-microglobulin in the methylotropic yeast, Pichia pastoris. The recombinant β2-microglobulin formed amyloid fibrils as demonstrated by electron microscopy and atomic force microscopy. With fluorescence microscopy, we observed the amyloid fibrils and its extension process.
3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclear NMR at the residue level. We showed that the modification of the buried thiol group of β-lactoglobulin destabilizes the entire parts of the molecule.
4. An early refolding intermediate of β-lactoglobulin is known to contain non-native α-helical structure. The early stage of β-lactoglobulin refolding was studied using ultra-rapid mixing techniques combined with hydrogen exchange labeling proved by heteronuclear NMR.We demonstrated that, in the kinetic intermediate accumulated at 2 msec of refolding, the non-native α-helix is formed at the N-terminal region of the molecule.
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