Mechanism of G protein Activation by Ric-8A

描述(申请人提供)：将进行研究，以了解G蛋白是如何由一种名为Ric-8A的蛋白质因子在细胞质中激活的。异三聚体G蛋白调节细胞代谢、分泌、电导、基因转录、细胞分裂和细胞运动，因此对人类所属的真核生物领域的生命是必不可少的。G蛋白的错误调节与癌症和其他一系列与普通医学相关的疾病有关。虽然大多数由异三聚体G蛋白控制的过程发生在细胞膜上，但最近的研究表明，Gα亚单位(G？)也控制细胞质中的某些事件。其中重要的一点是细胞的不对称分裂，这对胚胎发育至关重要。RIC-8A是G？在这个过程中。RIC-8A是一种鸟嘌呤核苷酸交换因子，催化G？活性部位的鸟苷二磷酸(GDP)交换为鸟苷三磷酸(GTP)。该反应使G？变为所谓的“激活”状态。在初步研究中，首席调查员的实验室证明了Ric-8A诱导和稳定G？i1(Gi-Class G？亚单位)处于结构不均匀或熔融的球状状态。无核苷酸的Ric-8A：G？I1中间体在没有GTP的情况下是稳定的。需要检验的假设是，Ric-8A通过改变G？I1的全局和局部结构，通过增加灵活性和诱导动态行为来催化核苷酸交换。该项目的总体目标是表征从Ric-8A与G？I1 GDP结合到形成无核苷酸的Ric-8A：G？I1复合体的交换反应的轨迹。该提案的前两个目标分别涉及Ric-8A：G？I1复合体的结构和动态行为。第一个目的是用双电子-电子共振(DER)谱观察G？I_1：RIC-8A络合物形成时G？I_1和Ric-8A结构的大范围变化。氢-氚交换，随后的蛋白质分解和质谱学(HDXMS)将被用来观察接近单一氨基酸残基的细节水平的结构变化。在第二个目标中，将进行单个分子的Forster共振能量转移(SmFRET)研究，无论是自由扩散的还是固定在固体基质上的，以确定G？I1：RIC-8A复合体的形成是否伴随着任何一种蛋白质的动态行为的变化。该提案的第三个目的是了解Ric-8A在从GDP结合状态向无核苷酸状态转变过程中改变G？I1结构的机制。将使用鹿和高密度X射线质谱仪相结合的方法来研究这种转变。定点定向自旋标记和电子顺磁共振谱结合突变扫描实验将被用来确定对G？I1结合和全球环境基金活性至关重要的Ric-8A残基，以及Ric-8A与G？I1接触的位置。

DESCRIPTION (provided by applicant): Research will be conducted to understand how G proteins are activated in the cytoplasm of the cell by a protein factor called Ric-8A. Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to life in the doman of eukaryotes to which humans belong. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (G?) also control certain events in cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of G? in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that catalyzes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of G?. This reaction transitions G? to the so-called "activated" state. In preliminary studies, the Principal Investigator's laboratory demonstrated that Ric-8A induces and stabilizes G?i1 (a Gi-class G? subunit) in a structurally heterogeneous or molten globule-like state. The nucleotide-free Ric-8A:G?i1 intermediate is stable in the absence of GTP. The hypothesis to be tested is that Ric-8A catalyzes nucleotide exchange by altering the global and local structure of G?i1, and by increasing flexibility and inducing dynamic behavior. The overall goal of the project is to characterize the trajectory of the exchange reaction from binding of Ric-8A to G?i1.GDP, to the formation of the nucleotide-free Ric-8A:G?i1 complex. The first two aims of the proposal address, respectively, the structure and the dynamic behavior of the Ric- 8A:G?i1 complex. The first aim is to use Double Electron-Electron Resonance (DEER) spectroscopy to observe large-scale changes in the structures of G?i1 and Ric-8A upon formation of the G?i1:Ric-8A complex. Hydrogen-Deuterium eXchange, followed by proteolysis and Mass Spectrometry (HDXMS) will be employed to observe structural changes at a level of detail that approaches single amino acid residues. In the second aim, Forster Resonance Energy Transfer (smFRET) studies of individual molecules, either freely diffusing or immobilized on a solid matrix, will be conducted to determine whether formation of the G?i1:Ric-8A complex is accompanied by changes in the dynamic behavior of either protein. The third aim of the proposal is to understand the mechanism by which Ric-8A alters the structure of G?i1 in the transition from the GDP- bound state to the nucleotide-free state. A combination of DEER and HDXMS methods will be used to study this transition. Site Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy, together with mutational scanning experiments, will be used to identify Ric-8A residues that are critical to G?i1-binding and GEF activity, and the sites at which Ric-8A makes contact with G?i1.