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Mechanism of G protein alpha subunit activation by receptors

受体激活G蛋白α亚基的机制

基本信息

批准号：
09680645
负责人：
WAKAMATSU Kaori
金额：
$ 1.98万
依托单位：
GUNMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680645/
关键词：
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

项目摘要

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

为了阐明G蛋白信号转导的详细机制，我们通过G蛋白-受体相互作用的生化和理化分析获得了以下结果：1． Gi1alpha 结合的 mastoparan-X 的构象：我们开发了一种泛素融合蛋白系统，可以轻松制备用稳定同位素均匀标记的肽。通过在由此获得的用^ 13 C和^ 15 N标记的mastoparan-X的Gi1alpha存在下进行TRNOE分析，获得与Gi1alpha结合的mastoparan-X的精确构象，其主链rmsd值小至0.33_.2。牛脑G蛋白β亚基的新型一步亲和纯化：通过以Gi1α亚基为配体的新型亲和层析，可以一步纯化牛脑β亚基。这一新方案预计对于从组织来源制备βγ亚基非常有用。3．受体 mi 激活后 Gi1alpha 的构象变化 ... 更多 metics ：为了研究配体受体刺激增强 G 蛋白 GDP 释放的机制，通过 CD 分析了受体模拟物引起的 Gi1alpha 二级结构变化。发现 Gi1alpha 的 α 螺旋含量与其激活平行减少。 4。受体激活Gi1α的机制：Gi1α氨基酸序列的二级结构预测表明，受体结合域中的α5螺旋在被受体模拟物激活后解开。 α5 螺旋通过由高度保守的 Asp 和 Lys 残基形成的离子对连接到 beta2/beta3 环，鸟嘌呤结合位点位于 beta2 链的 N 末端。这表明α5螺旋中的构象变化通过离子对诱导GDP结合位点的另一种构象变化，最终增强结合的GDP的释放。与这一假设一致，缺乏离子对的突变蛋白被发现很难被m2-毒蕈碱受体激活。人们发现离子对对于 GDP 与 α 亚基的紧密结合也很重要。较少的