STRUCTURAL STUDIES OF G PROTEIN-COUPLED RECEPTORS

描述（由申请人提供）：G蛋白偶联受体（GPCR）在生物医学中很重要，因为它们是约50%市售药物的靶点。对于这些受体在分子或原子上的结构和功能，我们知之甚少。GPCR的唯一三维X射线晶体学模型是处于非活性构象的基态视紫红质。视紫红质是视网膜中的光敏感蛋白，作为一种典型的GPCR已被广泛研究。整合膜蛋白含有7个跨膜螺旋，其为其11-顺式-视网膜发色团提供结合位点。光子的吸收引发发色团构象的变化，然后引发受体三级结构的变化。这导致受体的细胞质表面发生改变，从而允许与其同源G蛋白转导素（Gt）结合。这引发了信号转导过程中的进一步步骤。大多数GPCR以配体的形式响应分子信号。特定GPCR与特定配体的结合导致配体特异性细胞反应。大多数GPCR的配体结合位点与视紫红质中的视网膜口袋一致。配体的结合引起与视紫红质中光子吸收相同种类的构象变化，并且对于所有GPCR，信号转导的其余分子机制是相似的。本提案的组成部分将增进我们对气相化学还原反应结构和功能的了解。首先，激活的GPCRS的寡聚状态将通过探测在不同洗涤剂条件下分离的视紫红质的四级结构的实验来解决。将对这些制剂进行生理功能评估。该项目的第二部分将使用单分子力显微镜来探测膜与视紫红质或5-羟色胺5 HT 1AR受体之间的相互作用，以了解GPCR的动力学和稳定性。活化视紫红质的晶体学研究是本计画的第三部分。该项目的最后一个组成部分要求进一步努力纯化transformin/视紫红质复合物的生化和结构表征。这些项目都为一类重要的蛋白质提供了结构信息，这类蛋白质提供了广泛的实验和理论挑战。蛋白质家族对人类健康的重要性使这项努力值得。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are important in biomedicine because they are the targets of about 50% of commercially available drugs. Only little is known about the structure and function of these receptors in any molecular or atomic detail. The only three-dimensional X-ray crystallographic model for a GPCR is that of ground-state rhodopsin in an inactive conformation. Rhodopsin, the light-sensing protein in retina, has been extensively studied as a prototypical GPCR. The integral membrane protein contains seven trans- membrane helices that provide a binding site for its 11-cis-retinal chromophore. Absorption of a photon triggers a change in the chromophore's conformation and then in the receptor's tertiary structure. This results in alterations on the receptor's cytoplasmic surface that permit binding of transducin (Gt), its cognate G- protein. This initiates further steps in the signal transduction process. Most GPCRs respond to molecular signals in the form of ligands. Binding of specific ligands by specific GPCRs results in a ligand-specific cellular response. The ligand binding site for most GPCRs coincides with the retinal pocket in rhodopsin. Binding of a ligand causes the same kinds of conformational changes as does absorption of a photon in rhodopsin, and the remaining molecular mechanisms for signal transduction are similar for all GPCRs. Our understanding of GPCR structure and function will be increased by components of this proposal. First, the oligomeric state of activated GPCRS will be addressed by experiments probing the quaternary structure of rhodopsin isolated under varying detergent condtions. Assessment of physiological function will be made for these preparations. The second part of the project will use single-molecule force microscopy to probe the interactions between the membrane and rhodopsin or the serotonin 5HT1AR receptor to understand the dynamics and stabilities of GPCRs. Crystallographic studies of activated rhodopsin make up the third part of the project. The last component of the project calls for further efforts in purifying the transducin/rhodopsin complex for biochemical and structural characterization. These projects all provide structural information for an important class of proteins, a class that provides extensive experimental and theoretical challenges. The importance of the protein family for human health makes this effort worthwhile.