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1990 Fiscal Year Final Research Report Summary

Studies on the Mechanism of Physiological Function of Hemoglobin by Using Artificial Mutants

Research Project

Project/Area Number 01570045
Research Category

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

Allocation TypeSingle-year Grants
Research Field General physiology
Research InstitutionOsaka University

Principal Investigator

IMAI Kiyohiro  Osaka Univ. Med. Sch., Dept. of Physicochem. Physiol., Assoc. Prof., 医学部, 助教授 (50028528)

Co-Investigator(Kenkyū-buntansha) ISHIMORI Koichiro  Kyoto Univ., Fac. of Eng., Div. of Mol. Eng., Res. Assoc., 工学部, 助手 (20192487)
MIYAZAKI Gentaro  Osaka Univ., Fac. of Eng. Sci., Dept. of Biophys. Eng., Res. Assoc., 基礎工学部, 教務職員 (50166146)
WATANABE Manabu  Osaka Univ. Med. Sch., Dept. of Physicochem. Physiol., Res. Assoc., 医学部, 助手 (30182950)
Project Period (FY) 1989 – 1990
KeywordsArtificial Mutants / Hemoglobin / Site-directed Mutagenesis / Protein Engineering / Amino Acid Substitution / Recombinant DNA / Allosteric Effect
Research Abstract

Artificial hemoglobin mutants which contained single amino acid substitutions at particular sites were synthesized by site-directed mutagenesis using recombinant DNA and their oxygen binding function, light absorption spectra, proton nuclear magnetic resonance (NMR) spectra and resonance Raman scattering were measured to explore the roles of amino acid residues which are considered to be a key residue for the allosteric properties of hemoglobin.
Four mutants were synthesized : Hb Y145betaF in which Phe substitutes for Tyr-145beta which is considered to induce a tertiary structure change in the beta subunit ; Hb W37betaF in which Phe substitutes for Trp-37beta which forms a hydrogen bond with Asp-94alpha at the alpha1-beta2 interface ; Hb H92betaV and Hb H92betaD in which the proximal His at 92beta is replaced by Val or Asp. The M13 Phase-E. coli system was used to introduce the mutations into human globin gene and to express the globin gene.
The changes in oxygen binding functions (oxygen affinity, the Bohr effect, co-operativity, effect of inositol hexaphosphate) of Hb Y145betaF were moderate while those of Hb W37betaF were drastic. The tertiary and quaternary structure data acquired from UV light absorption, NMR, and resonance Raman spectra were nearly consistent with the functional data. It was noted that the important role of Tyr-145beta is that it has a side chain whose size is appropriate to fit to the tyrosine pocket rather than that it forms a hydrogen bond with Asp-94beta. The drastic functional changes in Hb W37betaF may partly be attributed to partial dissociation into alphabeta dimers.
Hb H92betaV and Hb H92betaD showed drastic functional changes. The replacement of the proximal His by neutral or negatively charged residue caused disapearance of allosteric effects whereas the heme iron of the mutant chains were maintained in a ferrous state, different from the M-type hemoglobins.

  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] K.Imai: "Structural and functional consequences of amino acid substitutions in hemoglobin as manifested in natural and artificial mutants" Protein Sequence and Data Analysis. 2. 81-86 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Ishimori: "NMR study of human mutant hemoglobins synthesized in <Eschrichia>___ー <Coli>___ー.Consequences of α42(C7) tyrosine substitutions" Journal of Biological Chemistry. 264. 14624-14626 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 石森 浩一郎: "蛋白質工学におけるヘモグロビンの協同効果発現機序の研究:四次構造転移における特定水素結合の寄与" 生物物理. 29. S46- (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 石森 浩一郎: "蛋白質工学によるヘモグロビン機能の改変(2)" 生物物理. 29. S202- (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Imai: "Molecular mechanism of the physiological function of hemoglobin studied by using artificial mutants" Japanese Journal of Physiology. 40. S61- (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Imai: "Siteーdirected mutagenesis in haemoglobin:Functional role of tyrosineー42(C7)α at the α1ーβ2 interface" Journal of Molecular Biology. 219. (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 籏野 昌弘 編: "Protein structural Analysis,Folding and Design" 日本学会出版センタ-およびElsevier, 237 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 池原 森男 編: "Protein Engineering:Protein Design in Basic Research,Medicine,and Industry" 日本学会出版センタ-およびSpringerーVerlag, 355 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K. Imai et al.: "Structural and Functional Consequences of Amino Acid Substitutions." Prot. Seq. Data Anal.2(2). 81-86 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Ishimori et al.: "NMR Study of Human Mutant Hemoglobins Synthesized in Escherichia Coli. Consequences of alpha42(C7) Tyrosine Substitutions." J. Biol. Chem.264(25). 14624-14626 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Ishimori et al.: "Study of the Mechanism of Cooperative Effects in Hemoglobin by Protein Engineering : a Contribution of a particular hydrogen bond to quaternary structure transition." Seibutsubutsuri. 29(suppl.). 46 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] I. Ishimori et al.: "Alteration of Hemoglobin Function by Protein Engineering (2)" Seibutsubutsuri. 29(suppl.). 202 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Imai et al.: "Molecular Mechanism of the Physiological Function of Hemoglobin Studied by Using Artificial Mutants" Jap. J. Physiol. 40(suppl.). 61 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Imai et al.: "Site-directed Mutagenesis in Hemoglobin : Functional Role of Tyrosine-42(C7)Alpha at the Alpha1-beta2 interface" J. Mol. Biol.219. (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M. Hatano (ed.): "Protein Structural Analysis, Folding and Design" Japan Sci. Soc. Press & Elsevier. 185-195 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M. Ikehara (ed.): "Protein Engineering : Protein Design in Basic Research, Medicine, and Industry" Japan Sci. Soc. Press & Springer-Verlag. 213-218 (1990)

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

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Published: 1993-08-12  

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