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2003 Fiscal Year Final Research Report Summary

Sequence effects of new N-capping motif CPxP on structural stability of YhhP protein

Research Project

Project/Area Number 14572038
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionTokyo University of Pharmacy and Life Science

Principal Investigator

SHINDO Heisaburo  Tokyo University of Pharmacy and Life Science, School of Pharmacy, Professor, 薬学部, 教授 (80138966)

Project Period (FY) 2002 – 2003
KeywordsYhhp protein / Point mutant / NMR / CD-melting / Urea denaturation / Guanidine hydrochloride / Charge interaction / Helix dipole
Research Abstract

YhhP protein is implemented in cell division and possesses a common sequence motif, CPxP. In this research project is to exploit biological and structural roles of this motif in YhhP protein function. To this end, we constructed a series of point mutants at all positions of sequence LRCPEP including CPxP. Functional bioassay was performed for all mutant proteins and their stability against heat, guanidine hydrochloride and urea was monitored by CD at 222nm.
Research results were as follows. (1)Point mutant proteins C19S and P20A/P22A were completely defected and E21K was partially defected, and the rests of mutations did not result in significant defects. The structure of YhhP was not affected by C 19S mutation but strongly affected by double mutation of P20A/P22A. Thus, it was concluded that the CPxP motif is important both structurally and functionally. (2)According to the three-dimensional structure assessed by NMR, a1-helix was elongated by 2 residues in P20A/P22A, but it was shorten by one residue in P20A. Those results together with others indicated that LRCPEP indeed forms a new type of N-capping motif. (3)Mutations at the positions of Arg18 and Glu21 involved in changes in surface charge strongly affected protein stability, concluding that electrostatic interaction between Arg18 and Glu21 in YhhP is weak but that change of charges at individual residues destabilize significantly the protein in terms of melting point Tm and denaturation midpoints due to urea and guanidinehydrochloride. It was concluded that interaction of negative charge of Glu21 with the helix dipole is essential for stability of this protein YhhP.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] M.Tashiro, H.Shindo: "Identification of bound waters in the solution structure of Ribonuclease T1 using the double pulsed field gradient spin-echo NMR technique for selective water"Magn.Reson.Chem.. 40. 559-562 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Tashiro, H.Shindo: "NMR structure of ubiquitin-like domain in PARKIN : Gene product of familial Parkinson's disease"J.Biomolec.NMR. 25. 153-156 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Ono, O.Kusano, et al.: "The Linker Histone Homolog Hho 1p from Saccharomyces cerevisiae Represents a Winged Helix-turn-helix Fold as Determined by NMR Spectroscopy"Nucl.Acids Res.. 31. 7199-7207 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Shimizu et al.: "Yeast Ume6p Represser Permits Activator Binding but Restricts TBP Binding at the HOP1 Promoter"Nucl.Acids Res.. 31. 3033-3037 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Ono, O.Kusano, S.Shimotakahara-Tashiro, M.Shimizu, T.Yamazaki, H.Shindo: "The Linker Histone Homolog Hholp from Saccharomyces cerevisiae Represents a Winged Helix-turn-helix Fold as Determined by NMR Spectroscopy"Nucl.Acids Res.. 31. 3033-3037 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Shimizu, K.Takahashi, T.M.Lamb, H.Shindo, A.P.Mitchell: "Yeast Ume6p Repressor Permits Activator Binding but Restricts TBP Binding at the HOP1 Promoter"Nucl.Acids Res.. 31. 3033-3037 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Tashiro, S.Okubo, S.Tashiro, H.Hatanaka, H.Yasuda, M.Kainosho, S.Yokoyama, H.Shindo: "NMR Structure of Ubiquitin-like Domain in PARKIN : Gene Product of Familial Parkinson's Disease"J.Biomol.NMR. 25. 153-156 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Tashiro, K.Furihata, S.S.Tashiro, H.Shindo: "Identification of Bound Waters in the Solution Structure of Ribonuclease T_1 using the Double Pulsed Field Gradient Spin-echo NMR Technique for Selective Water Excitation"Magn.Reson.Chem.. 40. 559-562 (2002)

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

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Published: 2005-04-19  

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