STRUCTURAL STUDIES OF G-PROTEIN COUPLED RECEPTORS

G 蛋白偶联受体的结构研究

• 批准号: 6879356
• 负责人: KEVIN Donald RIDGE
• 金额: $ 16.32万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879356
• 关键词: G protein; X ray crystallography; membrane proteins; nuclear magnetic resonance spectroscopy; protein structure; receptor coupling; rhodopsin

DESCRIPTION (Applicant's Description): Integral membrane proteins pose a unique challenge to traditional methods of structure determination by virtue of the fact that they are present in a membrane. As a result, many researchers are reluctant to address the problems associated with membrane protein structure determination because of the extensive time commitment and the level of risk involved. More than half of the almost 500 targets for which the pharmaceutical industry has developed molecules that alleviate disease fall into a class of integral membrane proteins known as G-protein coupled receptors. Many of these receptors play key roles in cardiovascular disease, diabetes, hypertension, AIDS, and a variety of sensory and mental disorders. It is because of the significant difficulty and lack of established methodology for the expression, purification, and crystallization of G-protein coupled receptors that the development and application of additional, alternative approaches for their high-resolution structure determination is proposed. Specifically, the goals of this research are to provide solutions to the aforementioned concerns using the G-protein coupled receptor's rhodopsin and CCR5 as models. Recent observations from this and other laboratories on the remarkable propensity of G-protein coupled receptor fragments to fold and assemble in an autonomous manner suggests that the cytoplasmic, membrane-embedded, and extracellular regions of these receptors can be regarded as independent domains. Further, biochemical studies on various soluble polypeptide subdomains of the cytoplasmic surface of rhodopsin show that they effectively mimic the signaling functions of the activated receptor. Initial efforts will focus on the construction of molecular models for these receptors by combining high resolution structural information obtained through NMR or crystallographic analysis of sections of the surface and transmembrane domains along with distance constraints derived from crosslinking studies aimed at determining the nearest neighbor organization of the transmembrane spans. A three-part, multidisciplinary approach to solve the three-dimensional structures of the intact receptors by X-ray crystallography will also be investigated. This will involve the large-scale expression and purification of these receptors or their mutants, the use of antibody fragments or soluble protein targets as exogenous "hydrophilic domains" to promote receptor crystallization, and the analysis of bicontinuous lipidic cubic phases for their ability to provide a suitable hydrophobic environment for the nucleation and growth of well-ordered crystals. Collectively, it is anticipated that these approaches will provide a framework for determining high-resolution structures of entire families of membrane proteins and will ultimately allow a more rational approach to the design of drugs for these essential targets.

描述(申请人描述)：完整的膜蛋白构成一种独特的 对传统结构确定方法的挑战 它们存在于膜中这一事实。因此，许多研究人员正在 不愿解决与膜蛋白结构相关的问题 由于大量的时间投入和风险水平而做出的决定 牵涉其中。在药物治疗的近500个目标中，超过一半 工业界已经开发出减轻疾病的分子属于一类 整体膜蛋白称为G蛋白偶联受体。其中许多 受体在心血管疾病、糖尿病、高血压、 艾滋病，以及各种感觉和精神障碍。这是因为 表达的难度很大，缺乏既定的方法， G蛋白偶联受体的纯化和结晶 开发和应用其他替代方法，以实现其 提出了高分辨率的结构确定方法。具体地说， 本研究旨在通过使用 以G蛋白偶联受体视紫红质和CCR5为模型。最近的观察结果 从这个实验室和其他实验室得出的G蛋白的显著倾向 偶联受体片段以自主方式折叠和组装 提示细胞的胞质、膜包埋和胞外区域 这些受体可以被视为独立的结构域。此外，生物化学 黄瓜胞质表面不同可溶性多肽亚区的研究 视紫红质表明，它们有效地模拟了 激活的受体。最初的努力将集中在分子的构建上。 通过结合高分辨率结构信息建立这些受体的模型 通过对表面的部分进行核磁共振或晶体分析而获得的 和跨膜结构域以及由以下来源的距离约束 旨在确定的最近邻组织的交联研究 跨膜跨越。由三个部分组成的多学科方法来解决 用X射线结晶学研究完整受体的三维结构 也将接受调查。这将涉及到大规模的表达和 纯化这些受体或其突变体，使用抗体片段 或以可溶性蛋白质为靶标的外源“亲水结构域”促进 受体结晶和双连续脂立方相的分析 为他们提供合适的疏水环境 有序晶体的成核和生长。总体而言，预计 这些方法将提供一个框架来确定高分辨率 整个膜蛋白家族的结构，最终将允许 以更合理的方法设计针对这些关键靶点的药物。

项目成果

NMR analysis of rhodopsin-transducin interactions.

视紫质-转导蛋白相互作用的核磁共振分析。

• DOI: 10.1016/j.visres.2006.07.024
• 发表时间: 2006
• 期刊: Vision research
• 影响因子: 1.8
• 作者: Ridge,KD;Marino,JP;Ngo,T;Ramon,E;Brabazon,DM;Abdulaev,NG
• 通讯作者: Abdulaev,NG

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states.

异源三聚体 G 蛋白 α 亚基中与受体刺激的鸟嘌呤核苷酸交换相关的构象变化：GTPgammaS 结合状态的 NMR 分析。

• DOI: 10.1074/jbc.m509851200
• 发表时间: 2006
• 期刊: The Journal of biological chemistry
• 作者: Ridge,KevinD;Abdulaev,NajmoutinG;Zhang,Cheng;Ngo,Tony;Brabazon,DanielleM;Marino,JohnP
• 通讯作者: Marino,JohnP

Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor.

开发基于表面的跨膜蛋白检测：选择性固定功能性 CCR5（一种 G 蛋白偶联受体）。

• DOI: 10.1016/j.ab.2005.10.025
• 发表时间: 2006
• 期刊: Analytical biochemistry.
• 作者: Silin,VitaliiI;Karlik,EvanA;Ridge,KevinD;Vanderah,DavidJ
• 通讯作者: Vanderah,DavidJ