Deciphering GPCR signal transduction through NMR structure and dynamics studies

通过 NMR 结构和动力学研究破译 GPCR 信号转导

• 批准号: 8950825
• 负责人: Joshua James Ziarek
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8950825
• 关键词: Adrenergic Receptor; Agonist; Amino Acids; Applied Research; Area; Arrestins; Basic Science; Binding; Biochemical; Biochemistry; Boston; Cells; Chemicals; Chemistry; Chimeric Proteins; Codon Nucleotides; Communities; Complex; Comprehension; Data Analyses; Data Collection; Development; Diphosphates; Discipline; Disease; Environment; Escherichia coli; Faculty; Funding; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Gatekeeping; Gene Silencing; Guanosine Triphosphate; Health; Heterotrimeric GTP-Binding Proteins; Human; Insecta; Institutes; Isotope Labeling; Label; Laboratories; Laboratory Research; Lead; Length; Ligand Binding; Ligands; Marketing; Mass Spectrum Analysis; Measures; Membrane Proteins; Mentors; Methods; Molecular Biology; Monitor; Motion; Muramidase; Mutation; NMR Spectroscopy; Neurotensin; Neurotensin Receptors; Nucleotides; Outcome; Pattern; Pharmaceutical Preparations; Phase; Phosphopeptides; Phosphorylation; Phosphotransferases; Physics; Protein Biochemistry; Protein Family; Protein Kinase C; Proteins; Proteome; Public Health; Reaction; Receptor Activation; Research; Role; Scheme; Scientist; Serine; Signal Pathway; Signal Transduction; Social Sciences; Solutions; Specificity; Structure; Surface; System; Techniques; Technology; Testing; Threonine; Training; Translating; United States National Institutes of Health; Universities; Work; X-Ray Crystallography; analog; arrestin 1; arrestin 2; base; directed evolution; drug discovery; empowered; extracellular; flexibility; human disease; insight; interest; macromolecule; medical schools; member; milligram; millisecond; molecular recognition; nanobodies; nanodisk; nanosecond; professor; programs; protein complex; public health relevance; receptor; receptor structure function; receptor-mediated signaling; research study; sortase; structural biology; theories

 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.

 描述（由申请人提供）：G 蛋白偶联受体 (GPCR) 是关键的真核信号转导看门人，代表人类蛋白质组中最大的蛋白质家族，拥有 800 多个成员。近年来，X射线晶体学已经产生了许多G蛋白偶联受体结构，但变构信号传导机制仍不清楚。 GPCR 的 X 射线晶体学需要多种形式的受体稳定，从而限制构象动力学。研究：GPCR 的定向进化使得原核表达系统能够产生毫克量的功能性同位素标记蛋白质。该技术为 GPCR 与各种配体以及与 G 蛋白和视紫红质抑制蛋白复合物的 NMR 研究开辟了新领域。 PI 将重点关注神经降压素受体，利用 NMR 光谱来探测溶液中 GPCR 激活的结构和构象动力学。培训：拟议在哈佛医学院瓦格纳教授实验室进行的培训将巩固 PI 对 NMR 理论和实施的理解。除了先进的核磁共振技术外，PI 还将获得对膜蛋白生物化学、纳米圆盘和定向进化的透彻了解。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项目。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至关重要。特别是，瓦格纳博士是大分子数据收集和分析领域的领导者。哈佛医学院拥有近 8,000 名教职员工和 17 个附属设施。医学院的核心是其教育和研究项目。医学院设有基础学科和社会科学学科九个系。更广阔的波士顿/剑桥地区拥有众多研究实验室，包括麻省理工学院、怀特海德和博德研究所，对结构生物学、生物化学、分子生物学、化学和物理学感兴趣，科学家们对与该提案相关的一般主题感兴趣。对公众健康的影响：GPCR 介导的信号通路与多种人类疾病有关，而 GPCR 是目前市场上估计 30-40% 的药物的靶标。鉴于其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。