• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

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
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥23,500,000 (Direct Cost: ¥23,500,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1987: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1986: ¥18,000,000 (Direct Cost: ¥18,000,000)
KeywordsFolding Intermediate / Conformational Fluctuation / H<double arrow>D Exchange / Fast CD Measurement / Molten Globule / alpha-Lactalbumin / Tryptophan Synthase / Staphiloccocal Nuclease / モルテングビュ-ル / αーラクトアルブミン / リゾチーム / 中間変性状態 / ジスルフィド結合 / 牛αーラクトアルブミン / トリプトファン / D→H交換 / 三次構造のゆらぎ / 馬リゾチーム / Ca^<2+>結合 / 牛α-ラクトアルブミン / 熱変性(中間)状態 / 【Ca^(2+)】結合
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.

Report

(5 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • 1987 Annual Research Report
  • 1986 Annual Research Report
  • Research Products

    (40 results)

All Other

All Publications (40 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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [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
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Kuwajima,M.Jitani and S.Sugai: "Characterization of the Critical state in Protein Folding:Effects of Guanidine Hydrochloride and Specific Ca Binding on the Folding Kinetics of alpha-Lactalbumin" J.Molecular Biology. 206. 547-561 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Y.Harushima and S.Sugai: "Hydrogen Exchange of the Tryptophan Residues in Bovine,Goat,Guinea Pig,and Human alpha-Lactalbumin" Biochemistry. 28. 8568-8576 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Kuwajima: "The Molten Globule State as a Clue for Understanding Folding and Cooperativity of Globular Proteins" Proteins:Structure,Function and Genetics. 6. 87-103 (1989)

    • Related Report
      1989 Annual Research Report
  • [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 Alpha-Lactalbumin and Lysozyme" J.Inorganic Biochemistry. 37. 185-191 (1989)

    • Related Report
      1989 Annual Research Report
  • [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 beta_2 subunit is a “Molten Globule"" FEBS Letters. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] E.A.Permyakov,V.M.Grischenko,L.P.Kalinichenko,N.Y.Oriov,K.Kuwajima and S.Sugai: "Calcium-regulated Interaction of Human alpha-Lactalbumin with Bee Venom.Melittin" Biophysical.Chemistry. 35. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] S.Sugai,K.Nitta and H.Tsuge: "Second Forum on Peptides (Colloque Inserum Vol.174)" John Libby,Eurotext,London Paris, 612 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] S.Sugai,K.Kuwajima and K.Nitta: "Protein Structural Analysis,Folding and Design(ed.M.Hatano)" Japan Scientic Society Press,Elsevier, 237 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yoshihiaro,Harushima;Kunihiro,Kuwajima;Shintaro,Sugai: Biopolymers. 27. 629-644 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Katsutoshi,Nitta;Hideaki,Tsuge;Shintaro,Sugai;Keiichi,Shimazaki: Biological Chemistry,Hoppe Seyler. 369. 671-675 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kunihiro,Kuwajima;Atsushi,Sakuraoka;Syoichi.Fueki;Michio,Yoneyama;Shintaro,Sugai: Biochemistry. 27. 7419-7428 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Masamichi.Ikeguchi;Shintaro,Sugai: International Journal Peptide and Protein Research. 30. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kunihiro,Kuwajima;Masahiro,Mitani;Shintaro,Sugai: Journal Molecular Biology. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Katsutoshi,Nitta;Shintaro,Sugai: European Journal Biochemistry. (1989)

    • Related Report
      1988 Annual Research Report
  • [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, 24-44 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kunihiro Kuwajima: FEBS Letters. 221. 115-118 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Katsutoshi Nitta: FEBS Letters. 223. 405-408 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Yoshiaki Harushima: Biopolymers. 27. (1988)

    • Related Report
      1987 Annual Research Report
  • [Publications] Kunihiro kuwajima: Int.J.Peptide & Protein Research. 27. 18-27 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Masahiro Mitani: J.Biological Chemistry. 26. 8824-8829 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Masamichi Ikeguchi: J.Biochemistry(Tokyo). 99. 1191-1201 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Masaki Hamano: J.Biochemistry(Tokyo). 100. 1617-1622 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Masamichi Ikeguchi: Biochemistry. 25. 6965-6972 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] Yoshiaki Harushima: Biopolymers.

    • Related Report
      1986 Annual Research Report

URL: 

Published: 1987-03-31   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi