Deciphering GPCR signal transduction through NMR structure and dynamics studies

 描述（由申请人提供）：G蛋白偶联受体（GPCR）是真核细胞信号转导的关键守门人，代表了人类蛋白质组中最大的蛋白质家族，有800多个成员。近年来，X-射线晶体学已经获得了许多G蛋白偶联受体结构，但变构信号的机制仍然未知。GPCR的X射线晶体学需要多种形式的受体稳定化，其随后限制构象动力学。研究：GPCR的定向进化使得能够从原核表达系统产生毫克量的功能性同位素标记蛋白质。该技术为具有各种配体以及与G蛋白和抑制蛋白复合的GPCR的NMR研究开辟了领域。专注于神经降压素受体，PI将使用NMR光谱来探测溶液中GPCR活化的结构和构象动力学。培训：在哈佛医学院瓦格纳教授的实验室进行的拟议培训将巩固PI对NMR理论和实施的理解。除了先进的核磁共振技术，PI将获得膜蛋白生物化学，纳米盘和定向进化的透彻理解。总之，这些技术将使PI能够建立一个独立的，NIH资助的结构生物学研究计划。环境：哈佛大学医学院的格哈德瓦格纳教授的实验室为这个项目提供了最佳环境。他的工作对生物分子表征的多维NMR实验的发展不可或缺;特别是，瓦格纳博士是大分子数据收集和分析的领导者。哈佛医学院拥有近8,000名教师和17个附属设施。在医学院的核心是其教育和研究计划。医学院有九个系，分别是基础科学和社会科学。更广泛的波士顿/剑桥地区包含许多研究实验室，包括麻省理工学院，怀特黑德和布罗德研究所，对结构生物学，生物化学，分子生物学，化学和物理学感兴趣，科学家对与此提案相关的一般主题感兴趣。对公众健康的影响：GPCR介导的信号通路与许多人类疾病有关，并且GPCR是目前市场上估计30-40%的所有药物的靶标。鉴于它们在健康和疾病中的基本作用，详细了解GPCR的结构和功能对于对细胞信号传导和分子识别感兴趣的基础科学界以及对药物发现感兴趣的应用科学界都具有价值。

 DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are critical eukaryotic signal transduction gatekeepers and represent the largest protein family in the human proteome, with more than 800 members. In recent years, X-ray crystallography has yielded many G protein-coupled receptor structures but the mechanism of allosteric signaling remains unknown. X-ray crystallography of GPCRs requires multiple forms of receptor stabilization that, subsequently, limit conformational dynamics. RESEARCH: Directed evolution of GPCRs enables milligram quantities of functional, isotopically-labeled protein to be produced from prokaryotic expression systems. This technology opens the field for NMR studies of GPCRs with various ligands and in complex with G proteins and arrestins. Focusing on the neurotensin receptor, the PI will use NMR spectroscopy to probe the structure and conformational dynamics of GPCR activation in solution. TRAINING: The proposed training in Professor Wagner's laboratory at Harvard Medical School will solidify the PI's comprehension of NMR theory and implementation. In addition to advanced NMR techniques, the PI will acquire a thorough understanding of membrane protein biochemistry, nanodiscs, and directed evolution. Together, these techniques will empower the PI to establish an independent, NIH-funded structural biology research program. ENVIRONMENT: Professor Gerhard Wagner's laboratory at Harvard University Medical School presents the optimal environment for this project. His work has been integral to the development of multidimensional NMR experiments for biomolecule characterization; in particular, Dr. Wagner is a leader in data collection and analysis of large macromolecules. Harvard Medical School has nearly 8,000 faculty and 17 affiliated facilities. At the core of the Medical School are its educational and research programs. The Medical School has nine departments in basic and social science disciplines. The wider Boston/Cambridge area contains numerous research laboratories including MIT, Whitehead, and Broad Institutes with interests in structural biology, biochemistry, molecular biology, chemistry and physics, with scientists interested in the general themes related to this proposal. IMPACT ON PUBLIC HEALTH: GPCR-mediated signaling pathways have been related to numerous human diseases, and GPCRs are the targets of an estimated 30-40% of all drugs currently on the market. In view of their fundamental roles in health and disease, a detailed understanding of GPCR structure and function is of value to the basic science community interested in cell signaling and molecular recognition, as well as to the applied science community interested in drug discovery.