Mechanism of G protein alpha subunit activation by receptors

为了阐明G蛋白信号转导的详细机制，我们通过对G蛋白-受体相互作用的生化和物理化学分析，获得了以下结果：1。Gi 1 α结合的mastoparan-X的构象：我们开发了一种泛素融合蛋白系统，该系统能够方便地制备用稳定同位素均匀标记的肽。通过TRNOE分析，在Gi 1 α存在下，对由此获得的用^ C和^ N标记的乳晕蛋白-X<13><15>进行分析，获得了与Gi 1 α结合的乳晕蛋白-X的精确构象，其主链rmsd值小至0.33 - 0.2。一种新的牛脑G蛋白β γ亚基的一步亲和纯化方法：以Gi 1 α亚基为配基的亲和层析法，可以一步纯化牛脑β γ亚基。这一新的方法对于从组织来源中制备β-γ亚基是非常有用的。受体激动剂激活后Gi 1 α的构象变化 关于我们 metics：为了研究配体受体刺激增强G蛋白释放GDP的机制，通过CD分析了受体模拟物引起的Gi 1 α二级结构的变化，发现Gi 1 α的α-螺旋含量随着其激活而减少.受体激活Gi 1 α的机制：Gi 1 α氨基酸序列的二级结构预测表明，受体结合结构域中的α 5螺旋在受体模拟物激活后解旋。α 5螺旋通过高度保守的Asp和Lys残基形成的离子对与β 2/β 3环连接，鸟嘌呤结合位点位于β 2链的N端。这表明α 5螺旋的构象变化通过离子对诱导GDP结合位点的另一个构象变化，最终增强结合的GDP.In协议的释放与这一假设，突变蛋白，缺乏离子对被发现是由M2-毒蕈碱受体激活不良。发现离子对对于GDP与α亚基的紧密结合也是重要的。少

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