| Project/Area Number |
09044221
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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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) |
KUWATA Kazuo Gifu University, School of Medicine, Associate Prof., 医学部, 助教授 (00170142)
BATT Carl A. コーネル大学, 食品学部, 教授
CARL A. Batt Cornell University, Department of Food Science, Professor
片岡 幹雄 大阪大学, 大学院・理学研究科, 助教授 (30150254)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Protein / Denaturation / Protein folding / Yeast / beta-Lactoglobulin / NMR / alpha*beta transition / isotope labeled protein |
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| Research Abstract |
Elucidation of the mechanisms of protein folding, by which the genetic information contained in the primary amino acid sequence of a protein is transmitted to its unique three-dimensional structure, is essential for understanding the structure and function of proteins. beta-Lactoglobulin is an intriguing model for clarifying the mechanism of the alpha-helix to beta-sheet (alpha-beta) transition of proteins, a key issue for understanding the folding and biological function of a number of protein molecules. We carried out the International Scienlific Research Program in order to clarify the mechanism of beta-lactoglobulin folding and obtained the following results.
1. Bovine beta-lactoglobulin A has been expressed in the methylotropic yeast Pichia pastoris. In a fed-batch fermenter, after 72hr of methanol induction, the secreted protein reached levels of 1g./L.The physical characteristics of recombinant protein were indistinguishable from those of the native bovine protein.
2. The recombin
… More
ant bovine beta-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol (TEE) were characterized by ^1H, ^<13>3C, and ^<15>5N multidimensional NMR spectroscopy. Overall secondary structures in the native state were similar to those of the crystal structure. On the other hand, beta-lactoglobulin in the TEE state was composed of many alpha-helical segments.
3. We determined the solution structure in the native state based on heteronuclear NMR techniques. The overall structure of monomeric beta-lactoglobulin at pH 2.0 in aqueous solution is very similar to the X-ray structure obtained at pH 6.5.
4. We analyzed the folding kinetics of beta-lactoglobulin by NMR measurements coupled with the H/D) exchange reaction. The results suggest that, during refolding, the native-like core beta-sheet and several non-native helices are formed rapidly due to hydrophobic collapse, and subsequently, the remaining beta-sheet is "induced" through interaction with the preexisting core beta-sheet. Less
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