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Experimental Approach to Understanding the Principle of Protein Folding

Research Project

Project/Area Number 01044087
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
Research InstitutionInstitute for Protein Research, Oska University

Principal Investigator

YUTANI Katsuhide  Institute for Protein Research, Osaka University, たんぱく質研究所, 助教授 (90089889)

Co-Investigator(Kenkyū-buntansha) MILES Edith W.  National Institutes of Health, USA, チーフ
SUGINO Yoshinobu  Kansai Medical School, 教養部, 教授 (00028177)
TSUNASAWA Susumu  Institute for Protein Research, Osaka University, 蛋白質研究所, 助教授 (30029962)
Project Period (FY) 1989 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1991: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1990: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
KeywordsCalorimetry / Conformational Stability / Protein folding / Proline / Lysozyme / Tryptophan Synthase / Stability / Protein Folding / Trypyophan Synthase / Calorimetry / Proline / Mutant Protein / Denaturation
Research Abstract

We have studied mutant proteins in order to elucicate the role of amino acid residues in protein folding, protein stability, and enzymatic function. In this report, we describe the outline of two papers.
1. In order to elucidate the role of a proline residue in protein folding, the unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103. The refolding kinetics of the P71G/PI03G mutant were found to be similar to those of the wild type protein. Other mutants such as P103G or P71G, and A47P with its three prolines, gave identical slow refolding phases.
2. To understand how the alpha and beta_2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha_2beta_2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha_2beta_2 complexes that contain wild type alpha subunits or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha_2beta_2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Isothermal calorimetric titrations of wild type beta_2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.

Report

(2 results)
  • 1991 Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] K.Yutani,S.Hayashi,Y.Sugisaki,&K.Ogasahara: "Role of Conserved Proline Residues in Stabilizing Tryptophan Synthase α-Subunit:Analyzed by Mutants with Alanine or Glycine." Proteins,. 9. 90-98 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T.Hering,K.Yutani,Y.Taniyama,& M.Kikuchi: "Effect of Proline Mutations on the Unfolding and Refolding of Human Lysozyme:The slow Refolding Kinetic Phase Does not Results from Proline Cis-Trans Isomerization." Biochemistry. 30. 9882-9891 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Ogasahara,K.Hiraga,W.Ito,E.W.Miles,& K.Yutani: "Origin of the Mutual Activation of the α and β_2 Subunits in the α_2β_2 Complex of Tryptophan Synthase:Effect of Alanine or Glycine Substitutions at Proline Residues in the α Subunit" J.Biol.Chem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y.Taniyama,K.Ogasahara,K.Yutani,& M.Kikuchi: "Folding Mechanism of Mutant Human Lysozyme C77/95A with Increased Secretion Efficiency in Yeast." J.Biol.Chem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 油谷 克英: "蛋白質立体構造の構築原理は「アミノ酸置換の手法」でどこまで解明できたか" 「生物物理」. 32. 27-32 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 油谷 克英,中村 春木: "蛋白質工学" 朝倉書店、東京, 208 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Yutani, S. Hayashi, Y. Sugisaki, & K. Ogasahara: "Role of Conserved Proline Residues in Stabilizing Tryptophan Synthase alpha-Subunit : Analyzed by Mutants with Alanine or Glycine." Proteins. 9. 90-98 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Hering, K. Yutani, Y. Taniyama, & M. Kikuchi: "Effect of Proline Mutations on the Unfolding and Refolding of Human Lysozyme : The slow Refolding Kinetic Phase Does not Results from Proline Cis-Trans Isomerization." Biochemistry. 30. 9882-9891 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Ogasahara, K. Hiraga, W. Ito, E. W. Miles, & K. Yutani: "Origin of the Mutual Activation of the alpha and beta_2 Subunits in the alpha_2beta_2 Complex of Tryptophan Synthase : Effect of Alanine or Glycine Substitutions at Proline Residues in the alpha Subunit J. Biol. Chem." J. Biol. Chem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y. Taniyama, K. Ogasahara, K. Yutani, & M. Kikuchi: "Folding Mechanism of Mutant Human Lysozyme C77/95A with Increased Secretion Efficiency in Yeast." J, Biol. Chem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Yutani: "Recent Studies on Conformational Stability of a Protein and Protein folding with Mutant Proteins" Seibutubuturi (Japanese). 32. 27-32 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Yutani & H. Nakamura: Asakura-Shoten, Tokyo. Protein Engineering (Japanese), 208 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sugisaki,Y.,Ogasahar,K.,Miles,E.W.,& Yutani,K.: "Scanning Calorimetrie study of Tryptophan Synthase αーSubunit from E.coli,S.typhimurium and an Interspecies Hybrid" Thermochimica Acta. 163. 117-122 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yutani,K.,Hayashi,S.,Sugisaki,Y.,& Ogasahara,K.: "Role of Conserved Proline Residues in Stabilizing Tryptophan Synthse αーSubunit: Analysis by Mutants with Alanine or Glycine" PROTEINS Structure,Function,and Genetics. 9. 90-98 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Go,M.,Tomoda,T.,Honda,M.,& Yutani,K.: "Domain and Module Structure in α/β Barrel of Tryptophan Synthase α Subunit" PROTEINS Structure,Function,and Genetics.

    • Related Report
      1990 Annual Research Report
  • [Publications] 小笠原 京子,油谷 克英: "トリプトファン合成酵素の構造と機能" 蛋白質核酸酵素. 35. 201-211 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yutani,K.& Ogasahara,K.: "Protein Structural Analysis,Folding and Design(Edited by Hatano,M.)" JapanScientific Societies Press Elsevier, 237 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 油谷 克英(分担執筆): "新生化学実験講座第1巻タンパク質III高次講造" 東京化学同人(日本生化学会編), 436 (1990)

    • Related Report
      1990 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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