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1990 Fiscal Year Final Research Report Summary

Studies on the Critical Structure of Protein Folding by Means of Site-Directed Amino Acid Replacements.

Research Project

Project/Area Number 01580258
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionHokkaido University

Principal Investigator

KUWAJIMA Kunihiro  Hokkaido University, Department of Polymer Science, Instructor, 理学部, 助手 (70091444)

Project Period (FY) 1989 – 1990
KeywordsProtein Folding / Transition State / Staphylococcal Nuclease / Stopped-Flow CD / Site-Directed Mutation / Kinetics
Research Abstract

Investigations of the transition state of folding have primary importance in elucidating the mechanism of protein folding. Here, we have used staphylococcal nuclease (SNase) as a model protein for analysis of the critical structure in the transition state and established an effective expression system to utilize site-directed amino acid replacements for such an analysis. The following results were obtained. (1) Unfolding and refolding of wild-type SNase induced by concentration jumps of urea have been studied by stopped-flow CD measurements in the peptide region. An intermediate that has an appreciable amount of peptide secondary structure accumulates at an early stage of the refolding, but it has also been shown to be significantly less stable than similar intermediates previously observed in other proteins. (2) Effects of specific ligands (Ca^<2+> and pdTp) for SNase on the kinetic folding have been analyzed quantitatively to obtain an insight into the structure of the transition state. Comparison of the present results with those on the two other Ca^<2+>-binding proteins (alpha-lactalbumin and parvalbumin) in previous studies has led to the conclusion that the critical structure in the transition state reflects the hierarchical nature of protein structure folding. (3) An expression-secretion plasmid for SNase in E. Coli, which is useful for making SNase mutants, was constructed by use of a lac-tac tandem promoter followed by the ribosome-binding site and the signal sequence of E. Coli protease III. (4) A mutant SNase (P117G) in which Pro117 was replaced by glycine was made, and its kinetic unfolding and refolding were investigated to study the effect of proline cis-trans isomerism on the kinetics. The proline to glycine mutation simplifies the kinetics of both unfolding and refolding and has been shown to be useful for analysis of the transition state of folding.

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Kuwajima,K.: "Transient Intermediates in the Folding of Dihydrofolate Reductase as Detected by Far Ultraviolet Circular Dichroism Spectroscopy" Biochemistry.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Permyakov,E.A.: "CalciumーRegulated Interaction of Human αーLactalbumin with Bee Venom Melittin" Biophysical Chem.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sugawara,T.: "Folding of Staphylococcal Nuclease A Studied by Equilibrium and Kinetic Circular Dichroism Spectra" Biochemistry. 30. 2698-2706 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuwajima,K.: "Kinetics of DiトulfideーBond Reduction in αーLactalbumin by Dithiothreitol and Molecular Basis of Superreactivity of the Cys6ーCys120 Disulfide Bond" Biochemistry. 29. 8240-8249 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Goldberg,M.E.: "An Early Immunoreactive Folding Intermediate of the Tryptophan Synthase β_2Subunit Is a Molten Globule" FEBS Letters. 263. 51-56 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuwajima,K: "The Molten Globule State as a Clue for Understanding the Folding and Cooperativity of GlobularーProtein Structure" Proteins. 6. 87-103 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuwajuma, K., Garvey, E. P., Finn, B. E., Matthews, C. R., & Ssgat, S.: "Transient Intermediates in the Folding of Dihydrofolate Reductase as Detected by Far Ultraviolet Circular Dichroism Spectroscopy" Biochemistry.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Pcrmyakov, E. A., Grishchenko, V. M., Kalinichenko, L., Orlov, N. Y., Kuwajuma, K., & Sugai, S.: "Calcium-Regulated Interactions of Human alpha-Lactalbumin with Bee Venom Melittin" Biophysical Chem.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sugawara, T., Kuwajima, K., & Kugai, S.: "Folding of Staphylococcal Nuclease A Studied by Equilibrium and Kinetic Circular Dichroism Spectra" Biochemistry. 30. 2698-2706 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuwajima, K., Ikeguchi, M., Sugawara, T., Hiraoka, Y., & Sugai, S.: "Kinetics of Disulfide-Bond Reduction in alpha-Lactalbumin by Dithiothreitol and Molecular Basis of Superreactivity of the Cys6-Cys120 Disulfide Bond" Biochemistry. 29. 8240-8249 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Goldberg, M. E., Semisotnov, G. V., Friguet, B., Kuwajima, K., Ptitsyn, O. B., & Sugai, S.: "An Early Immunoreactive Folding Intermediate of the Tryptophan Synthase beta 2 Subunit Is a Molten Globule" FEBS Letters. 263. 51-56 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuwajima, K.: "The Molten Globule State as a Clue for Understanding the Folding and Cooperativity of Globular-Protein Structure" Proteins. 6. 87-103 (1989)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-08-12  

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