Mechanism of G protein Activation by Ric-8A

描述（由申请人提供）：将进行研究，以了解G蛋白如何在细胞的细胞质中被称为Ric-8A的蛋白质因子激活。异三聚体G蛋白调节细胞代谢、分泌、电导率、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至关重要。G蛋白的失调与癌症和一系列与普通医学相关的其他疾病有关。虽然由异源三聚体G蛋白控制的大多数过程发生在细胞膜上，但最近的研究表明，G α亚单位（G?）也控制细胞质中的某些事件。其中重要的是不对称细胞分裂，这对胚胎发育至关重要。ric8a是G？在这个过程中。Ric-8A是一种鸟嘌呤核苷酸交换因子（GEF），在G?的活性位点催化鸟苷二磷酸（GDP）交换鸟苷三磷酸（GTP）。这个反应转变为G？到所谓的“激活”状态。在初步研究中，首席研究员的实验室证明了ric8a诱导和稳定G？i （a G -class G?）亚基)在结构上不均匀的或熔融的球状状态。无核苷酸的ric8a:G？i1中间体在没有GTP的情况下是稳定的。要验证的假设是，ric8a通过改变G?的整体和局部结构来催化核苷酸交换。通过增加灵活性和诱导动态行为。该项目的总体目标是表征Ric-8A与g11结合的交换反应的轨迹。GDP，以形成无核苷酸的ric8a:G？i1复杂。该提案的前两个目标分别针对Ric- 8A:G？i1复杂。第一个目标是利用双电子-电子共振（DEER）光谱来观察G？i和ric8a在G？i1: Ric-8A复杂。氢-氘交换，然后是蛋白质水解和质谱（HDXMS）将被用来观察结构变化的细节水平，接近单个氨基酸残基。在第二个目标中，将对自由扩散或固定在固体基质上的单个分子进行福斯特共振能量转移（smFRET）研究，以确定G？1 . Ric-8A复合物伴随着两种蛋白的动态行为变化。提案的第三个目标是了解ric8a改变G？在从GDP结合状态到无核苷酸状态的过渡中。将采用DEER和HDXMS相结合的方法来研究这种转变。位点定向自旋标记和电子顺磁共振光谱以及突变扫描实验将用于鉴定对G？i - 1结合和GEF活性，以及Ric-8A与G - 1接触的位点。

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.