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1989 Fiscal Year Final Research Report Summary

Characterization of Structural Intermediates of Proteins by Hydrogen Exchange Method

Research Project

Project/Area Number 61420050
Research Category

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

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

Principal Investigator

SUGAI Shintaro  Hokkaido University, Faculty of Science Professor, 理学部, 教授 (80000727)

Co-Investigator(Kenkyū-buntansha) KUWAJIMA Kunihiro  Hokkaido University, Faculty of Science, Assistant, 理学部, 助手 (70091444)
NITTA Katsutoshi  Hokkaido University, Faculty of Science, Instructor, 理学部, 講師 (80001858)
Project Period (FY) 1986 – 1989
KeywordsFolding Intermediate / Conformational Fluctuation / H<double arrow>D Exchange / Fast CD Measurement / Molten Globule / alpha-Lactalbumin / Tryptophan Synthase / Staphiloccocal Nuclease
Research Abstract

To characterize the intermediates on the pathway of unfolding or folding of proteins, the isotope exchange and technique was used for alpha-lactalbumin in the intermediate states the results were compared with those in the native state. Two of four tryptophan residues exchanged the NH protons fast with small activation energy of exchange, and others very slowly with large activation energy in the native state. One and two dimensional ^1H-NMR studies of bovine, goat, human and guinea pig alpha-lactalbmin showed that the latter residues are Trp 26 and 60, which exchange the protons after exposure on the molecular surface due to the conformational fluctuation. In the acid state, the NH protons in the side chain and those in main chain exchange fast, but in two phases. The protons in the fast phase are exposed almost on the surface, however the others are included in interior of the molecule to some extent. The activation energies of exchange mean the considerable conformational fluctuation of the acid (and also folding) intermediate. The isotope exchange and pH-jump methods showed conservation of the secondary structure region in the acid intermediate in the native state.
Tryptophan synthase beta_2 subunit from E.coli has been shown to assume a molten globular folding intermediate with a hydrophobic fluorescence dye. We examined such a fact in the fast CD spectra in the aromatic and peptide regions. Also, the folding intermediate of staphilococcal nuclease was characterized by use of the fast CD and isotope exchange methods.

  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] Kunihiro Kuwajima,Hidetoshi Yamaya,Sakae Miwa and Shintaro Sugai: "Rapid Formation of the Secondary Structure Framework in Protein Folding Studied by Stopped-flow Circular Dichroism" FEBS Letters. 221. 115-118 (1987)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Katsutoshi Nitta, Hideaki Tsuge,Keichi Shimazaki and Shintaro Sugai: "Calcium-Binding Lysozymes" Biological Chem.Hoppe Seyler. 369. 671-675 (1988)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kunihiro Kuwajima,Masahiro Mitani and Shintaro Sugai: "Characterization of the Critical State in Protein Folding Effects of Guanidine Hydrochloride and specific Ca^<2+> Binding on the Folding Kinetics of α-Lactalbumin" J.Mol.Biology. 206. 547-561 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Katsutoshi Nitta and Shintaro Sugai: "The Evolution of Lysozyme and α-Lactalbumin" Eur.J.Biochemistry. 182. 111-118 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] J.Desmet,H.Van Dael,F.Van Cauwelaert,K.Nitta and S.Sugai: "Comparison of the Binding of Ca^<2+> and Mn^<2+> to Bovine α-Lactalbumin and Lysozyme" J.Inorganic Biochemistry. 37. 185-191 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.E.Goldberger,G.V.Semisotonov,B.Friguet,K.Kuwajima,O.B.Ptitsyn and S.Sugai: "An Early Immunoreactive Folding Intermediate of the Tryptophan Synthase β_2 subunit is a “Molten Globule"" FEBS Letter. inpress (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 須貝新太郎: "生命現象と生化学(八木康一、石井信一編)" 北大図書刊行会, (1988)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shintaro Sugai,Kunihiro Kuwajima and Katsutoshi Nitta: "Protein Structural Analysis,Folding and Design(ed.M.Hatano)" Japan Scientic Society Press Elsevier, 53-73 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kunihiro Kuwajima, Hidetoshi Yamaya, Sakae Miwa and Shintaro Sugai: "Rapid Formation of the Secondary Structure Framework in Protein Folding Studied by Stopped-flow Circular Dichroism" FEBS Letters. 221. 115-118 ((1987))

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katsutoshi Nitta, Hideaki Tsuge, Keiichi Shimazaki and Shintaro Sugai: "Calcium-Binding Lysozymes" Biological Chemistry, Hoppe Seyler 369, 671-675 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kunihiro Kuwajima, Masahiro Mitani and Shintaro Sugai: "Characterization of the Critical State in Protein Folding: Effects of Guanidine Hydrochloride and Specific Ca^<2+> Binding on the Folding Kinetics of alpha-Lactalbumin" J.Molecular Biology 206, 247-561 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katsutoshi Nitta and Shintaro Sugai: "The Evolution of Lysozyme and alpha-Lactalbumin" European J. Biochemistry 182, 111-118 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] J.Desmet, H.Van Dael, F.Van Cauwelaert, K.Nitta and Shintaro Sugai: "Comparison of the Binding of Ca^<2+> and Mn^<2+> to Bovine alpha-Lactalbumin and Lysozyme" J. Inorganic Biochemistry 37, 185-191 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.E.Goldberger, G.V.Semisotonov, B.Friguet, K.Kuwajima, O.B.Ptitsyn and Shintaro Sugai: "An Early Immunireactive Folding Intermediate of the Tryptophan Synthase beta_2 Subunit is a "Molten Globule"" FEBS Letters (1990).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shintaro Sugai: "Seimei Gensho to Seikagaku (eds. K.Yagi and S.Ishii)" Hokkaido University Press (1988) Chap. 3.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shintaro Sugai, Kunihiro Kuwajims and Katsutoshi Nitta: "Protein Structural Analysis, Folding and Design (ed. M.Hatano)" Japan Scientic Society Press Elsevier (1990) p.53-73.

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

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Published: 1993-03-26  

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