Chimeric Proteins to Enhance Crystal Formation of G Protein Coupled Receptors (RM

描述（由申请人提供）：增强G蛋白偶联受体晶体形成的嵌合蛋白。本R21提案是对《膜蛋白生产和结构测定计划公告》（RFA编号：RFA-RM-04-026）的回应。该提案的总体目标是开发一种通用方法，以获得可用于基于结构的药物开发的G蛋白偶联受体（GPCR）的高分辨率结构。大多数激素和神经递质通过GPCR向细胞传递信息。GPCR代表了人类基因组中最大的膜蛋白家族，也是药物开发的最大靶点。我们建议使用人类β 2 AR作为模型系统，以开发一种通过增加其稳定性和亲水性（极性）表面积来生成GPCR的衍射质量晶体的方法。这将通过产生结构整合的嵌合蛋白来实现，其中可溶性蛋白通过替换第三胞内环（ICL 3）和羧基末端而整合到GPCR中。这种方法将解决GPCR晶体产生中的两个重要障碍：（1）GPCR的ICL 3和羧基末端是大多数GPCR中最动态（柔性）和非结构化的结构域。这些结构域将被高度结构化的同质蛋白质取代。三个剪接位点将确保可溶性蛋白质和GPCR之间的刚性结构偶联。(2)嵌合体的可溶性蛋白质组分将显著增加可用于晶格接触的极性表面积。如果β 2AR成功，这种方法可能会推广到其他GPCR。具体目标包括： 目标1.使用分子建模工具来鉴定可与β 2AR融合以产生稳定的功能性嵌合体的可溶性蛋白质。 目标2.产生、表达和优化融合蛋白。 简单总结。本研究的目的是开发一种通用的方法来获得G蛋白偶联受体（GPCRs）的高分辨率结构。这些结构应该有助于GPCR的药物发现过程，GPCR是人类基因组中最大的膜蛋白家族。作用于GPCR的药物可以对广泛的疾病产生影响，包括：心血管疾病、肺部疾病、炎症、糖尿病和肥胖症、行为障碍和阿尔茨海默病。

DESCRIPTION (provided by applicant): Chimeric Proteins To Enhance Crystal Formation Of G Protein Coupled Receptors. This R21 proposal is in response to the Program Announcement on Membrane Protein Production and Structure Determination (RFA Number: RFA-RM-04-026). The overall goal of this proposal is to develop a general approach to obtain high-resolution structures of G protein coupled receptors (GPCRs) that can be used for structure-based drug development. The majority of hormones and neuretransmitters communicate information to cells via GPCRs. GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug development. We propose to use the human beta2AR as a model system to develop a method for generating diffraction quality crystals of GPCRs by increasing their stability and their hydrophilic (polar) surface area. This will be accomplished by generating structurally integrated chimeric proteins in which a soluble protein is integrated into a GPCR by replacing the third intracellular loop (ICL3) and the carboxyl terminus. This approach will address two important obstacles in the generation of GPCR crystals: (1) The ICL3 and carboxyl termini of GPCRs are the most dynamic (flexible) and unstructured domains in most GPCRs. These domains will be replaced by a highly structured homogeneous protein. The three splice sites will ensure a rigid structural coupling between the soluble protein and the GPCR. (2) The soluble protein component of the chimera will significantly increase the polar surface area available for crystal lattice contacts. If successful with the beta2AR, this approach will likely be generalizable to other GPCRs. Specific aims include: Aim 1. Use molecular modeling tools to identify soluble proteins that can be fused to the beta2AR to generate stable, functional chimeras. Aim 2. Generate, express and optimize fusion proteins. Lay summary. The goal of this proposal is to develop a general method to obtain high-resolution structures of G protein coupled receptors (GPCRs). These structures should facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.