STRUCTURAL AND FUNCTIONAL DESCRIPTIONS OF G-PROTEIN COUPLED NEURORECEPTOR

G 蛋白偶联神经感受器的结构和功能描述

• 批准号: 8364095
• 负责人: Anthony Watts
• 金额: $ 0.08万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364095
• 关键词: Agonist; Binding; Collaborations; Colon Carcinoma; Coupled; Data; Data Analyses; Elements; Engineering; Escherichia coli; Funding; GTP-Binding Proteins; Grant; Human; Ligand Binding; Lipids; Membrane; Methods; Modeling; Modification; National Center for Research Resources; Neurologic; Neurotensin Receptors; Parkinson Disease; Personal Satisfaction; Positioning Attribute; Principal Investigator; Proteins; Reporter; Research; Research Infrastructure; Resolution; Resources; Sensory; Sensory Receptors; Signal Transduction; Source; Specificity; Spin Labels; Structure; Technology; Time; Translating; United States National Institutes of Health; Work; analytical method; computer generated; cost; design; dietary control; programs; protein activation; quantum; receptor; reconstitution

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our current research is focused on functional descriptions of a membrane-embedded, well characterized G-protein coupled neuroreceptor (GPCR) for which no high resolution structure is available. This receptor, the neurotensin receptor type 11 (NTS1; MW ~ 40kDa) is implicated in Parkinson's, dietary control and is a marker in colon cancer  GPCRs are a major focus for all major pharma in view of their importance in all sensory and neurological control but lack of structures and functional descriptions, preclude rational design of new therapies, despite their potential for contributing to human well-being and wealth creation. In part of this wider program of research, the Watts group is using ESR spin label methods to: i) determine structural constraints and secondary structure elements for NTS1; ii) characterize the conformational changes in NTS1 on agonist binding; iii) determine any specific lipid protein interactions required for ligand binding; iv) collate the structural and dynamic data accumulated into structural and functional models for GPCR signaling; and, v) understand how lipid specificity for NTS1 may be coupled to G-protein activation. NTS1 is expressed by us in E. coli, and we can manipulate cys residues and at the same time retain ligand binding capacity and G-protein activation  the wild type protein has 9 cys residues, four of which are buried in the receptor and not accessible to modification. The ESR methods the Watts group are using rely on DEER and spin-exchange for longer and shorter range distance methods respectively, with nitroxides at well-defined positions (from computer-generated models) at the end of trans-membrane helices of the receptor into which we have engineered reporter cys residues. Our collaboration with ACERT could help is in (i) providing much higher than X-band sensitivity for this work in which receptor in reconstituted, function-supporting membranes, is limiting (nM max); (ii) enable double quantum filtering (DQF) as an approach for resolving distances in conformationally sensitive NTS1 upon ligand binding (iii) input for proper analysis of data using various analytical methods appropriate to the data; (iv) translate the ESR data into structural information using CNS, developed at ACERT.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们目前的研究重点是膜嵌入的、特征明确的 G 蛋白偶联神经受体 (GPCR) 的功能描述，但目前尚无高分辨率结构。这种受体，即神经降压素受体 11 型（NTS1；MW ~ 40kDa）与帕金森病、饮食控制有关，并且是结肠癌的标志物。GPCR 是所有主要制药公司的主要关注点，因为它们在所有感觉和神经控制中都很重要，但缺乏结构和功能描述，妨碍了新疗法的合理设计，尽管它们有促进人类福祉和创造财富的潜力。在这个更广泛的研究计划中，Watts 小组正在使用 ESR 自旋标记方法来： i) 确定 NTS1 的结构约束和二级结构元素； ii) 表征激动剂结合时 NTS1 的构象变化； iii) 确定配体结合所需的任何特定脂质蛋白相互作用； iv) 将积累的结构和动态数据整理成 GPCR 信号传导的结构和功能模型； v) 了解 NTS1 的脂质特异性如何与 G 蛋白激活相结合。 NTS1是我们在大肠杆菌中表达的，我们可以操纵cys残基，同时保留配体结合能力和G蛋白激活能力，野生型蛋白有9个cys残基，其中4个埋在受体中，无法修饰。 Watts 小组使用的 ESR 方法依赖于 DEER 和自旋交换，分别用于较长距离和较短距离的方法，其中硝基氧位于受体跨膜螺旋末端明确的位置（来自计算机生成的模型），我们在其中设计了报告器 cys 残基。我们与 ACERT 的合作可以帮助：(i) 为这项工作提供远高于 X 波段的灵敏度，其中重构的功能支持膜中的受体受到限制（nM max）； (ii) 启用双量子过滤 (DQF) 作为解决配体结合时构象敏感 NTS1 距离的方法 (iii) 使用适合数据的各种分析方法输入数据进行正确分析； (iv) 使用 ACERT 开发的 CNS 将 ESR 数据转换为结构信息。