Structural and Biochemical Studies of the Metarhodopsin II-Transducin Complex

描述（由申请人提供）：G蛋白偶联受体（GPCR）通过检测细胞外信号并将其穿过细胞膜传递至细胞内鸟嘌呤核苷酸结合蛋白（G蛋白）来调节广泛的生理过程。然而，我们对信号转导的理解受到缺乏完全活化受体的高分辨率结构的限制，无论是单独的还是与G蛋白复合的。本研究的长期目标是使用视紫红质（视网膜的昏暗光感应受体）作为模型GPCR来解析这些结构。这些结构的确定将确定构象触发，促进G蛋白活化和G蛋白特异性。为了达到既定目标，我将解决以下具体目标：1。活性态视紫红质晶体结构的测定。为了形成活化的受体，我将使用组成型活性视紫红质突变体（N2 C/E113 Q/D282 C）。受体将在哺乳动物细胞中表达，并使用免疫亲和色谱法纯化。纯化的受体将结晶使用气相扩散技术和衍射数据将收集在同步加速器X射线光束线。2.红视紫红质-G蛋白复合物的生物化学表征。我将使用纯化的N2 C/E113 Q/D282 C突变体视蛋白和天然来源的G蛋白，通过修改视紫红质免疫亲和程序分离活性复合物。我将描述这个复杂的化学计量，稳定性和拆卸方面。这些信息将有助于回答有关视紫红质-转导素复合物领域的问题。这些研究也将指导配合物的结晶。3.确定视紫红质-G蛋白复合物的晶体结构。纯化的N2 C/E113 Q/D282 C muplant-G蛋白复合物将通过气相扩散结晶，并在同步加速器X射线光束线处获得衍射数据。将使用数据分析软件解决结构问题。获得受体-G蛋白复合物的结构将通过提高我们识别和治疗与不受调节的受体活性相关的疾病的能力来影响公共卫生的几个方面。这类疾病包括肥胖症和静止性夜盲症，每种疾病都是由受体蛋白突变引起的，这些突变导致受体活性发生变化。高分辨率的结构信息将有助于用专门定制的小分子来促进或阻碍受体活性来治疗这些疾病。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) regulate a wide range of physiological processes by detecting and transmitting an extracellular signal across a cell membrane to intracellular guanine-nucleotide binding proteins, G proteins. However, our understanding of signal transduction is limited by the absence of high resolution structures of the fully-activated receptor, alone and in complex with G protein. The long-term goal of this study is to resolve these structures using rhodopsin, the dim-light sensing receptor of the retina, as a model GPCR. Determination of these structures will define the conformational triggers that promote G protein activation and G protein specificity. To reach the stated goals I will address the following specific aims: 1. Determine the Crystal Structure of Rhodopsin in the Active State. To form activated receptors I will use a constitutively-active rhodopsin mutant (N2C/E113Q/D282C). Receptors will be expressed in mammalian cells and purified using immunoaffinity chromatography. Purified receptors will be crystallized using vapor-diffusion techniques and diffraction data will be collected at a synchrotron X-ray beamline. 2. Biochemically Characterize the Rhodopsin-G protein Complex. I will isolate the active complex by a modification of the rhodopsin immunoaffinity procedure using purified N2C/E113Q/D282C mutant opsin and G protein from natural sources. I will characterize this complex in regards to stoichiometry, stability, and disassembly. This information will be instrumental in answering questions in the field regarding the rhodopsin-transducin complex. These studies will also guide the crystallization of the complex. 3. Determine the Crystal Structure of the rhodopsin-G protein Complex. Purified N2C/E113Q/D282C mutant-G protein complexes will be crystallized by vapor diffusion and diffraction data will be obtained at a synchrotron X-ray beamline. Structures will be solved using data analysis software. PUBLIC HEALTH RELEVANCE Acquiring the structure of the receptor-G protein complex will impact several aspects of public health by improving our ability to recognize and treat disorders linked to unregulated receptor activity. The spectrum of such diseases includes obesity and stationary night blindness, each caused by mutations in the receptor protein that lead to changes in receptor activity. Treatment for these disorders with small molecules specifically tailored to promote or impede receptor activity will be facilitated by high-resolution structural information.