1993 Fiscal Year Final Research Report Summary
Kinetic Studies of Protein Folding Using Protein Engineering
| Project/Area Number |
03453170
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 | University of Tokyo (1992-1993)
Hokkaido University (1991) |
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Principal Investigator |
KUWAJIMA Kunihiro University of Tokyo, Graduate School of Science, Associate Professor, 大学院・理学系研究科, 助教授 (70091444)
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| Co-Investigator(Kenkyū-buntansha) |
IKURA Teikichi University of Tokyo, Graduate School of Science, Research Associate, 大学院・理学系研究科, 助手 (50251393)
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| Project Period (FY) |
1991 – 1993
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| Keywords | Protein Folding / Site-Directed Mutagenesis / Nuclease / alpha-Lactalbumin / Kinetics / Proline Isomerization / Molten Globule State |
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| Research Abstract |
In this study, staphylococcal nuclease and goat alpha-lactalbumin cloned and expressed by plasmids of Escherichia coli were used to investigate the mechanism of protein folding. Using various site-directed mutants of these proteins, stability of the molten globule state and kinetics of refolding were analyzed. The following results were obtained.
(1) In order to investigate effect of proline isomerization on the refolding kinetics, two proline mutants, Pro117Gly and Pro56Ala, of nuclease were constructed. (2) The mutants of nuclease are known to be classified into two classes : Class I mutants stabilize and class II mutants destabilize the molten globule state. The proteins with the class I (Val66Leu, Gly88Val and their double mutant) and class II (Ala90Ser, Ala69Thr and their double mutant) mutations were purified from Escherichia coli that has the respective mutant plasmids. The unfolding transition of each mutant protein was measured by CD spectroscopy in the peptide region. As expected, the secondary structure of the class I mutants is more stable than that of the wild-type protein, while that of the class II mutants is less stable. (3) The mutations (Ala30Ile, Ala30Thr and Thr33Ile) that change the hydrophobicity of the core of the molecule have been introduced in alpha-lactalbumin, and the effect of the mutations on the stability of the molten globule state was investigated by CD spectroscopy. The Ala30Ile and Thr33Ile mutations that increase the core hydrophobicity increase the stability of the molten globule, while the Ala30Thr mutation that decreases the hydrophobicity decreases the stability of the molten globule.
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