| Project/Area Number |
09308023
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | HOKKAIDO UNIVERSITY (1999)
Tokyo Metropolitan Organization for Medical Research (1997-1998) |
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Principal Investigator |
INAGAKI Fuyuhiko Grad. School of Pharm., Hokkaido Univ., Prof., 大学院・薬学研究科, 教授 (70011757)
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| Co-Investigator(Kenkyū-buntansha) |
OGURA Kenji Grad. School of Pharm., Hokkaido Univ., Inst., 大学院・薬学研究科, 助手 (50270682)
寺澤 宏明 財団法人東京都臨床医学総合研究所, 生理活性物質研究部門, 研究員 (10300956)
畠中 秀樹 (財)東京都臨床医学総合研究所, 生理活性物質研究部門, 研究員 (00260331)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥33,600,000 (Direct Cost: ¥33,600,000)
Fiscal Year 1999: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1998: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1997: ¥23,400,000 (Direct Cost: ¥23,400,000)
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| Keywords | Grb2 domain engineering / Neutrophil / TPR motif / SH3 / P67ィイD1phoxィエD1 / P47ィイD1phoxィエD1 / Rac / p47^<phox> / Rac / NMR / 緩和時間 / リンカー / フレキシビリティー / Vav / シス-トランス異性 / Ash / X線結晶構造解析 / X線小角散乱 / SH2 / 動的構造 |
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| Research Abstract |
We applied domain engineering to Ash/Grb2. We swapped nSH3 and cSH3 domains in Grb2 (SH3-SH2-SH3) and analyzed binding proteins using cell lysate from bovine brain. The binding proteins were similar to those for wild type Grb2, showing that each SH3 domain of Grb2 is independent . We also studied the phagocyte oxidation system in neutrophil. Phagocyte oxidation system consists of membrane bound gp91ィイD1phoxィエD1, p22phox and intracellular factor of p48ィイD1phoxィエD1, p67phox and p40ィイD1phoxィエD1. These intracellular factors have domain structure, comprising of SH3, PB1, PB2, TPR and PC motifs. We studied three dimensional structure of TRP motif in p67phox. Single crystal is now obtained and data collection has jut started using synchoroton radiationfacility.
Phagocyte oxidation. System is triggered by binding of Rac(GTP) to TPR motif and p47ィイD1phoxィエD1 SH3 SH3 domain to p22ィイD1phoxィエD1 PRR. We designed novel signaling protein which consists of TPR motif directly connected to the SH3 domain of p47phox. The activity of this protein was studied in cell-free system containing membrane fraction of neutrophil and Rac-GTP form. The designed protein is quite active to generate super oxide. Thus, we have succeeded in preparing a protein based on the result of structural biology. We are going to extend this technique to prepare novel signaling proteins and regulate signal transduction system using the designed proteins.
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