| Project/Area Number |
14572038
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
SHINDO Heisaburo Tokyo University of Pharmacy and Life Science, School of Pharmacy, Professor, 薬学部, 教授 (80138966)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Yhhp protein / Point mutant / NMR / CD-melting / Urea denaturation / Guanidine hydrochloride / Charge interaction / Helix dipole / CD melting / 尿素 / 塩酸グアニジン / YhhP蛋白質 / 点変異体 / N-キャッピング |
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| 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.
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