1989 Fiscal Year Final Research Report Summary
Characterization of Structural Intermediates of Proteins by Hydrogen Exchange Method
| Project/Area Number |
61420050
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Hokkaido University |
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Principal Investigator |
SUGAI Shintaro Hokkaido University, Faculty of Science Professor, 理学部, 教授 (80000727)
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| Co-Investigator(Kenkyū-buntansha) |
KUWAJIMA Kunihiro Hokkaido University, Faculty of Science, Assistant, 理学部, 助手 (70091444)
NITTA Katsutoshi Hokkaido University, Faculty of Science, Instructor, 理学部, 講師 (80001858)
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| Project Period (FY) |
1986 – 1989
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| Keywords | Folding Intermediate / Conformational Fluctuation / H<double arrow>D Exchange / Fast CD Measurement / Molten Globule / alpha-Lactalbumin / Tryptophan Synthase / Staphiloccocal Nuclease |
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| Research Abstract |
To characterize the intermediates on the pathway of unfolding or folding of proteins, the isotope exchange and technique was used for alpha-lactalbumin in the intermediate states the results were compared with those in the native state. Two of four tryptophan residues exchanged the NH protons fast with small activation energy of exchange, and others very slowly with large activation energy in the native state. One and two dimensional ^1H-NMR studies of bovine, goat, human and guinea pig alpha-lactalbmin showed that the latter residues are Trp 26 and 60, which exchange the protons after exposure on the molecular surface due to the conformational fluctuation. In the acid state, the NH protons in the side chain and those in main chain exchange fast, but in two phases. The protons in the fast phase are exposed almost on the surface, however the others are included in interior of the molecule to some extent. The activation energies of exchange mean the considerable conformational fluctuation of the acid (and also folding) intermediate. The isotope exchange and pH-jump methods showed conservation of the secondary structure region in the acid intermediate in the native state.
Tryptophan synthase beta_2 subunit from E.coli has been shown to assume a molten globular folding intermediate with a hydrophobic fluorescence dye. We examined such a fact in the fast CD spectra in the aromatic and peptide regions. Also, the folding intermediate of staphilococcal nuclease was characterized by use of the fast CD and isotope exchange methods.
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