STRUCTURAL STUDIES OF G-PROTEIN COUPLED RECEPTORS

G 蛋白偶联受体的结构研究

• 批准号: 6498231
• 负责人: KEVIN Donald RIDGE
• 金额: $ 22.2万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498231
• 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（申请人的描述）：整合膜蛋白构成独特的生物学功能。 对传统结构测定方法的挑战， 事实上，它们存在于膜中。因此，许多研究人员 不愿意解决与膜蛋白结构相关的问题 由于投入大量时间和风险水平， 涉案超过一半的近500个目标， 工业界开发出的缓解疾病的分子属于一类 被称为G蛋白偶联受体的整合膜蛋白。许多这些 受体在心血管疾病、糖尿病、高血压 艾滋病，以及各种感官和精神障碍。正是因为 严重困难和缺乏既定的表达方法， G蛋白偶联受体的纯化和结晶， 发展和应用其他替代办法， 提出了高分辨率结构测定。具体而言， 这项研究是为了提供解决上述问题的方法， 以G蛋白偶联受体的视紫红质和CCR5为模型。最近的观察 从这个实验室到其他实验室， 偶联受体片段以自主方式折叠和组装 表明，细胞质，膜包埋，和细胞外区域的 这些受体可以被认为是独立的结构域。此外，生物化学 研究了大肠杆菌胞质表面的各种可溶性多肽亚结构域， 视紫红质表明，它们有效地模拟了 激活受体初步的努力将集中在分子结构的构建上， 通过结合高分辨率结构信息， 通过核磁共振或晶体学分析获得的表面部分 和跨膜结构域沿着，距离限制来自 交联研究旨在确定最近邻组织的 跨膜跨度。一个由三部分组成的多学科方法来解决 通过X射线晶体学的完整受体的三维结构 也将受到调查。这将涉及大规模的表达和 这些受体或其突变体的纯化、抗体片段的使用 或可溶性蛋白靶作为外源性“亲水结构域”， 受体结晶和双连续立方相的分析 因为它们能够提供合适的疏水环境， 有序晶体的成核和生长。总的来说，预计 这些方法将为确定高分辨率 整个膜蛋白家族的结构，并最终允许 更合理的方法来设计针对这些基本目标的药物。