Deciphering GPCR signal transduction through NMR structure and dynamics studies

 描述(申请人提供)：G蛋白偶联受体(GPCRs)是关键的真核信号转导守门人，代表着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射线结晶学已经发现了许多与G蛋白偶联的受体结构，但变构信号的机制尚不清楚。GPCRs的X射线结晶学需要多种形式的受体稳定，从而限制构象动力学。研究：GPCRs的定向进化使原核表达系统能够产生毫克量的功能性、同位素标记的蛋白质。这项技术为具有各种配体的GPCRs以及与G蛋白和阻滞剂形成的复合体的核磁共振研究开辟了新的领域。PI将以神经降压素受体为重点，使用核磁共振波谱来探索溶液中GPCR激活的结构和构象动力学。培训：拟在哈佛医学院瓦格纳教授的实验室进行的培训将巩固PI对核磁共振理论和实施的理解。除了先进的核磁共振技术，PI还将对膜蛋白生物化学、纳米盘和定向进化有深入的了解。总而言之，这些技术将使PI能够建立一个独立的、由NIH资助的结构生物学研究计划。环境：哈佛大学医学院格哈德·瓦格纳教授的实验室为该项目提供了最佳环境。他的工作对于生物分子表征的多维核磁共振实验的发展是不可或缺的；尤其是，瓦格纳博士是大分子数据收集和分析方面的领导者。哈佛医学院有近8000名教职员工和17个附属机构。医学院的核心是它的教育和研究项目。医学院在基础学科和社会科学学科中有九个系。更广泛的波士顿/剑桥地区包括许多研究实验室，包括麻省理工学院、怀特黑德研究所和布罗德研究所，他们对结构生物学、生物化学、分子生物学、化学和物理感兴趣，科学家们对与这一提议相关的一般主题感兴趣。对公众健康的影响：GPCRs介导的信号通路与许多人类疾病有关，目前市场上估计有30%-40%的药物是GPCRs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是有价值的。

 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.