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.Gi1α结合的Mastoparan-X的构象：我们开发了一个泛素融合蛋白系统，可以方便地制备稳定同位素标记的多肽。通过TRNOE分析，得到了与Gi1α结合的MASTARAN-X的精确构象，主链RMSD值为0.33_2。一种新的一步亲和纯化牛脑G蛋白Betagamma亚基的方法：以Gi1α亚基为配基的亲和层析可一步纯化牛脑Betagamma亚基。这一新的方案有望在从组织来源中制备Betagamma亚基方面非常有用。受体mi…激活后Gi1α的构象变化更多的方法：为了研究配体受体刺激促进G蛋白释放GDP的机制，用CD分析了受体模拟引起的Gi1α的二级结构变化，发现Gi1α的α螺旋含量随着其激活而减少。受体激活Gi1α的机制：对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被解开。α5螺旋通过保守的Asp和Lys残基形成的离子对连接到Beta2/Beta3环上，在Beta2链的N端有一个鸟嘌呤结合位点。这表明，α5螺旋上的构象变化通过离子对诱导GDP结合部位的另一次构象变化，最终促进结合的GDP的释放。与这一假说一致，缺乏离子对的突变蛋白被发现不能被M2-M受体激活。研究发现，离子对对GDP与α亚基的紧密结合也很重要。较少

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