| Project/Area Number |
09680645
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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 |
Biophysics
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| Research Institution | GUNMA UNIVERSITY |
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Principal Investigator |
WAKAMATSU Kaori Gunma University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (40222426)
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| Co-Investigator(Kenkyū-buntansha) |
KOHNO Toshiyuki Mitsubishi Kasei Institute for Life Sciences, Dept.Structure Analysis, Researche, 構造解析研究室, 研究員
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | peptide / expression system / stable isotope / CD / NMR / TRNOE / affinity chromatography / conformation / G蛋白質 / マストパラン / compound 48 / 80 / 活性化 / レセプター / 突然変異体 / βγサブユニット / Compound48 / 二次構造変化 |
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| Research Abstract |
To elucidate detailed mechanisms of signal transduction via G proteins, we obtained the following results by biochemical and physicochemical analyses on G protein-receptor interactions :
1. Conformation of Gi1alpha-bound mastoparan-X : We developed a ubiquitin fusion protein system that enables facile preparation of peptides uniformly labeled with stable isotopes. By TRNOE analysis in the presence of Gi1alpha of the mastoparan-X labeled with ^<13> C and ^<15>N thus obtained, a precise conformation of mastoparan-X bound to Gi1alpha was obtained with the backbone rmsd value as small as 0.33 _.
2. A novel one-step affinity purification of the G protein betagamma subunits from bovine brain : By the novel affinity chromatography with Gi1alpha subunits as a ligand, bovine brain betagamma subunits could be purified in one step. This new protocol is expected to be quite useful for preparing betagamma subunits from tissue sources.
3. Conformational change of Gi1alpha upon activation by receptor mi
… More
metics : To investigate the mechanism whereby stimulation by a liganded receptor enhances the GDP release from G protein, secondary structure change of Gi1alpha caused by receptor mimetics was analyzed by CD.The alpha-helix content of Gi1alpha was found to decrease in parallel with its activation.
4. Mechanism of Gi1alpha activation by receptors : Secondary structure prediction of the amino acid sequence of Gi1alpha suggested that the alpha5 helix in the receptor binding domain is unwound upon activation by receptor-mimetics. The alpha5 helix is linked to the beta2/beta3 loop via the ion-pair formed by well-conserved Asp and Lys residues, and a guanine binding site is located to the N-terminus of the beta2 strand. This suggests that the conformation change in the alpha5 helix induces another conformation change in the GDP binding site via the ion-pair, to ultimately enhance the release of bound GDP.In agreement with this hypothesis, a mutant protein that lacks the ion-pair was found to be poorly activated by m2-muscarinic receptor. The ion-pair was found to be important also for the tight binding of GDP to the alpha subunit. Less
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