| Project/Area Number |
15380229
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
HIROSE Masaaki Kyoto University, The Graduate School of Agriculture, Professor, 農学研究科, 教授 (60026523)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Nobuyuki Kyoto University, The Graduate School of Agriculture, Instructor, 農学研究科, 助手 (20252520)
MIZUTANI Kimihiko Kyoto University, The Graduate School of Agriculture, Instructor, 農学研究科, 助手 (40314281)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2004: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2003: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | ovalbumin / serpin / loop insertion / conformation change / protease inhibition / タンパク質工学 / 卵白タンパク質 / セリンプロティナーゼ |
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| Research Abstract |
Ovalbumin is a member of serpin superfamily, but it does not have any proteinase inhibitor activity. We have done site-directed mutagenesis approach to confer serpin function on ovalbumin. We found by X-ray crystallography that an ovalbumin mutant R339T has an ability to undergo a serpin loop insertion after the P1-P1' cleavage by elastase. This was an important finding for the achievement of our aim, but the mutant did still not have an inhibitory activity against a protease. For further mutagenesis approach, we created a different ovalbumin mutant R339T/A352R in which the P1-P1' site is accessible against trypsin. Utilizing the mutant, a reliable HPLC analysis for the determination of the loop insertion rate was established. Because of the structural and functional situations of serpin, increased loop insertion rate should lead to the acquisition of the inhibitory activity. We therefore did further mutagenesis to accelerate the loop insertion rate on the basis of the data of crystal structure. Alternative mutants, K290T/A352R/R339T and R104/A352R/R339t, and the disulfide-reduced form of A352R/R339T, respectively, displayed 1.5, 3.7, and 6.7- fold increase in the loop insertion rate as compared A352R/R339T control. The mutation and disulfide reduction should give a more flexible nature on the distal sheet A structure. We therefore concluded that the transition state of the serpin loop insertion reaction assumes an opened sheet A conformation.
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