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Kinetic Studies of Protein Folding Using Protein Engineering

Research Project

Project/Area Number 03453170
Research Category

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

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionUniversity of Tokyo (1992-1993)
Hokkaido University (1991)

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)

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)
Project Period (FY) 1991 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1991: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsProtein Folding / Site-Directed Mutagenesis / Nuclease / alpha-Lactalbumin / Kinetics / Proline Isomerization / Molten Globule State / プロソン異性化反応 / タンパク質の構造形成 / ラクトアルブミン / タンパク質フォ-ルディング / ヌクレア-ゼ
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.

Report

(4 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Kuwajima,K.,Okayama,N.,Yamamoto,K.,Ishihara,T.,& Sugai,: "The Pro117 to Glycine Mutation of Staphylococcal Nuclease Simolifies the Unfolding-Folding Kinetics" FEBS Letters. 290. 135-138 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima,K.,Garvey E.P.,Finn,B.E.,Matthews,C.R.,Sugai: "Transient Intermediates in the Folding of Dihydrofolate Reductase as Derected by Far Ultraviolet Circular Dichroism Spectroscopy" Biochemistry. 30. 7693-7703 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima,K.: "Protein Folding in vitro" Current Opinion Biotech.3. 462-467 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yutani,K.,Ogasahara,K.,Kuwajima,K.: "Absence of the Thermal Transition in apo-α-Lactalbumin in the Molten Globule State" J.Mol.Biol.228. 347-350 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ikeguchi,M.,Sugai,S.,Fujino,M.,Kuwajima,K.: "Contribution of the 6-120 Disulfide Bond of α-Lactalbumin to the Stabilities of Its Native and Molten Globule States" Biochemistry. 31. 12695-12700 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima,K.,et al.: "Secondary Structure of Globular Proteins at the Early and the Final Stages in Protein Folding" FEBS Letters. 334. 265-268 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima, K., Okayama, N., Yamamoto, K., Ishihara, T., Sugai, S.: "The Pro117 to Glycine Mutation of Staphylococcal Nuclease Simplifies the Unfolding-Folding Kinetics" FEBS Letters. 290. 135-138 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima, K., Garvey, E.P., Finn, B.E., Matthews, C.R., Sugai, S.: "Transient Intermediates in the Folding of Dihydrofolate Reductase as Detected by Far Ultraviolet Circular Dichroism Spectroscopy" Biochemistry. 30. 7693-7703 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima, K.: "Protein Folding in vitro" Current Opinion Biotech.3. 462-467 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yutani, K., Ogasahara, K., Kuwajima, K.: "Absence of the Thermal Transition in apo-alpha-Lactalbumin in the Molten Globule State" J.Mol.Biol.228. 347-350 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ikeguchi, M., Sugai, S., Fujino, M., Kuwajima, K.: "Contribution of the 6-120 Disulfide Bond of alpha-Lactalbumin to the Stabilities of Its Native and Molten Globule States" Biochemistry. 31. 12695-12700 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima, K., Semisotnov, G.V., Finkelstein, A.V., Ptitsyn, O.B.: "Secondary Structure of Globular Proteins at the Early and the Final Stages in Protein Folding" FEBS Letters. 334. 265-268 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kuwajima,K.: "Secondary Structure of Globular Proteins at the Early and the Final stages in Protein Folding" FEBS Lett.334. 265-268 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kuwajima,K.: "Spectroscopic Techniques to Study Protein Folding c.Circular Dichroism" Methods in Molewlar Biology. (発表予定). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 桑島邦博・伊倉貞吉: "蛋白質の折れたたみ機構" 蛋白質・核酸・酵素. (発表予定). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 桑島邦博: "ストレス蛋白質の基礎と臨床,(一部執筆)" 中外医学社出版,永田和宏編集(発表予定), (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kuwajima,K.: "Protein folding in vitro" Current Opinion Biotech.3. 462-467 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Yutani,K.,Ogasahara,K. Kuwajima,K.: "Absence of the thermal transition in apo-alpha-lactalbumin in the molten globule state" J.MOL.Biol.228. 347-350 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Ikeguchi,M.,Sugai,S.,Fujino,M.,Kuwajima,K.: "Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states" Biochemistry. 31. 12695-12700 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 桑島 邦博: "蛋白質フォールディングの中間体としてのモルテン・グロビュール状態" 生物物理. 33. 26-30 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 桑島 邦博: "タンパク質の折れたたみ" 科学. 63. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kuwajima,K.,Okayama,Yamamoto,K.,Ishihara,T.,& Sugai,S.: "The Pro117 to Glycine Mutation of Staphylococcal Nuclease Simplifies the UnfoldingーFolding Kinetics" FEBS Letters. 290. 135-138 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kuwajima,K.,Garvey,E.P.,Finn,B.E.,Matthews,C.R.,Sugai: "Transient Intermediates in the Folding of Dihydrofolate Reductase as Detected by Far Ultraviolet Circular Dichroism Spectroscopy" Biochemistry. 30. 7693-7703 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Sugawara,T.,Kuwajima K.,& Sugai,S.: "Folding of Staphylococcal Nuclease A Studied by Equilibrium and Kinetic Circular Dichroism Spectra" Biochemistry. 30. 2698-2706 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Permyakov,E.A.,Grishchenko,V.M. et al.: "Calciumーregulated Interaction of Human αーLactalbumin with Bee Venom Melittin" Biophysical Chemistry. 39. 111-117 (1991)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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