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Role of α-β transition in the folding of proteins

Research Project

Project/Area Number 11694208
Research Category

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

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionOsaka University

Principal Investigator

GOTO Yuji  Osaka University, Institute for Protein Research, Professor, たんぱく質研究所, 教授 (40153770)

Co-Investigator(Kenkyū-buntansha) HOSHINO Masaru  Osaka University, Institute for Protein Research, Research Assistant, たんぱく質研究所, 助手 (70304053)
KAWATA Yasushi  Tottori University, Faculty of Engineering, Professor, 工学部, 教授 (40177697)
KUWATA Kazuo  Gifu University, School of Medicine, Associate Professor, 医学部, 助教授 (00170142)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
KeywordsProtein / Protein Folding / Heteronuclear NMR / β-Lactoglobulin / β2-Microglobulin / Fluorescence / Single Molecule Analysis / Amyloid Fibril / 変性
Research Abstract

The α-helix to β-sheet(α→β)transition of proteins is a key issue to understand the folding and biological function of a number of proteins. Folding of β-lactoglobulin is a useful model for clarifying the mechanism of the α→β transition because the kinetic intermediate contains non-native α-helical structure. With recombinant bovine β-lactoglobulin A uniformly labeled with ^<15>N and heteronuclear NMR spectroscopy, we analyzed its folding kinetics and dynamics.
1. The H/D exchange experiments have been performed on the native state, demonstrating the presence of a stable hydrophobic core.
2. To define the structural and dynamic properties of an early folding intermediate in β-lactoglobulin, the kinetics of folding was measured, using ultra-rapid mixing techniques in conjunction with hydrogen exchange labeling probed by heteronuclear NMR.We demonstrated that, in the early kinetic intermediate, the non-native α-helix is formed at the N-terminal region of the molecule.
3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclar NMR.We showed that the modification of the buried thiol group of β2-microglobulin destabilizes the entire parts of the molecule.
4. To understand the mechanism of amyloid formation, which is proposed to include the α→β transition, we expressed human β2-microglobulin in the methylotropic yeast, Pichia pastoris. The recombinant β2-microglobulin formed amyloid fibrils as demonstrated by electron microscopy and atomic force microscopy. With fluorescence microscopy, we observed the amyloid fibrils made of β2-microglobulin and its extension process.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Kuwata, Kazuo: "Solution structure and dynamics of bovine β-lactoglobuin A."Protein Science. 8(11). 2541-2545 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hong, Donpyo: "Clustering of fluorine-substituted alcohols as a factor responsible for their marked effects on proteins and peptides."Journal of American Chemical Society. 121(37). 8427-8433 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Forge, Vincent: "Is Folding of β-lactoglobulin non-hierarchic? Intermediate with native-like β-sheet and non native α-helix."Journal of Molecular Biology. 296(4). 1039-1051 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sakai, Kazuko: "Conformation and stability of thiol-modified bovine β-lactoglobulin."Protein Science. 9(10). 1719-1729 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kuwata, Kazuo: "Structural and kinetic characterization of early folding events in β-lactoglobulin."Nature Structural Biology. 8(2). 151-155 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kuwata, Kazuo: "High pressure NMR reveals a vatiety of fluctuating conformers in β-lactoglobulin."Journal of Molecular Biology. (印刷中). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 後藤祐児: "(訳本)タンパク質フォールディングのキネティクス、Bengt Nolting著"シュプリンガーフェアラーク東京. 180 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 後藤祐児・谷澤克行 編: "蛋白質の分子設計"共立出版. 200 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kuwata, K.: "Solution structure and dynamics of bovine β-lactoglobulin A."Protein Sci.. 8(11). 2541-2545 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hirota, N.: "Alcohol-induced denaturation of β-lactoglobulin : a close correlation to the alcohol-induced α-helix formation of melittin."Bioorg.& Med.Chem.. 7. 67-73 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hong, D.: "Clustering of fluorine-substituted alcohols as a factor responsible for their marked effects on proteins and peptides."J.Am.Chem.Soc.. 121(37). 8427-8433 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamasaki, R.: "Single molecular observation of the interaction of GroEL with substrate proteins."J.Mol.Biol.. 292(5). 965-972 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Forge, V.: "Is Folding of β-lactoglobulin non-hierarchic? Intermediate with native-like β-sheet and non-native α-helix."J.Mol.Biol.. 296(4). 1039-1051 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sakai, K.: "Conformation and stability of thiol-modified bovine β-lactoglobulin."Protein Sci.. 9(10). 1719-1729 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hoshino, M.: "High mobility of the phospholipid binding loop of human β2-glycoprotein I domain V revealed by heteronuclear NMR."J.Mol.Biol.. 304(5). 927-940 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kuwata, K.: "Structural and kinetic characterization of early folding events in β-lactoglobulin."Nature Struct.Biol.. 8. 151-155 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kuwata, K: "High pressure NMR reveals a variety of fluctuating conformers in β-lactoglobulin."J.Mol.Biol.. (in the press.).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sakai,Kazuko: "Conformation and stability of thiol-modified bovine β-lactoglobulin."Protein Science. 9(10). 1719-1729 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hoshino,Masaru: "High mobility of the phospholipid binding loop of human β2-glycoprotein I domain V revealed by heteronuclear NMR."Journal of Molecular Biology. 304(5). 927-940 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kuwata,Kazuo: "Structural and kinetic characterization of early folding events in β-lactoglobulin."Nature Structural Biology. 8(2). 151-155 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kuwata,Kazuo: "High pressure NMR reveals a variety of fluctuating conformers in β-lactoglobulin."Journal of Molecular Biology. (印刷中). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 後藤祐児: "(訳本)タンパク質フォールディングのキネティクス、Bengt Nolting著"シュプリンガーフェアラーク東京. 180 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 後藤祐児,谷澤克行 編: "蛋白質の分子設計"共立出版. 200 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kuwata,Kazuo: "Solution structure and dynamics of bovine β-lactoglobulin A"Protein Science. 8(12). 2541-2545 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Hoshino,Masaru: "Is folding of β-lactoglobulin non-hierarchic?"J.Mol.Biol.. 296(4). 1039-1051 (2000)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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