1998 Fiscal Year Final Research Report Summary
Folding Mechanism of beta-Lactogobulin
| 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
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| Project Period (FY) |
1997 – 1998
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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
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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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Research Products
(14 results)
