2000 Fiscal Year Final Research Report Summary
Role of α-β transition in the folding of proteins
| Project/Area Number |
11694208
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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) |
HOSHINO Masaru Osaka University, Institute for Protein Research, Research Assistant, たんぱく質研究所, 助手 (70304053)
KAWATA Yasushi Tottori University, Faculty of Engineering, Professor, 工学部, 教授 (40177697)
KUWATA Kazuo Gifu University, School of Medicine, Associate Professor, 医学部, 助教授 (00170142)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Protein / Protein Folding / Heteronuclear NMR / β-Lactoglobulin / β2-Microglobulin / Fluorescence / Single Molecule Analysis / Amyloid Fibril |
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| Research Abstract |
The α-helix to β-sheet(α→β)transition of proteins is a key issue to understand the folding and biological function of a number of proteins. Folding of β-lactoglobulin is a useful model for clarifying the mechanism of the α→β transition because the kinetic intermediate contains non-native α-helical structure. With recombinant bovine β-lactoglobulin A uniformly labeled with ^<15>N and heteronuclear NMR spectroscopy, we analyzed its folding kinetics and dynamics.
1. The H/D exchange experiments have been performed on the native state, demonstrating the presence of a stable hydrophobic core.
2. To define the structural and dynamic properties of an early folding intermediate in β-lactoglobulin, the kinetics of folding was measured, using ultra-rapid mixing techniques in conjunction with hydrogen exchange labeling probed by heteronuclear NMR.We demonstrated that, in the early kinetic intermediate, the non-native α-helix is formed at the N-terminal region of the molecule.
3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclar NMR.We showed that the modification of the buried thiol group of β2-microglobulin destabilizes the entire parts of the molecule.
4. To understand the mechanism of amyloid formation, which is proposed to include the α→β transition, 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 made of β2-microglobulin and its extension process.
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Research Products
(17 results)
