Production of 15N and 13C labeled GPCRs for NMR Spectroscopy

用于 NMR 波谱学的 15N 和 13C 标记 GPCR 的生产

• 批准号: 7478270
• 负责人: Brian K Kobilka
• 金额: $ 17.78万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7478270
• 关键词: Adenylate Cyclase; Adrenergic Receptor; Agonist; Alzheimer' s Disease; Baculoviruses; Behavior Disorders; Binding; Biochemical; Biological Models; Cabbage - dietary; Cardiovascular Diseases; Catecholamines; Complex; Coupling; Development; Diabetes Mellitus; Diet; Disease; Dopamine; Drug Interactions; Drug Receptors; Eating; Epinephrine; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Hormones; Human Genome; Inflammation; Insecta; Isotope Labeling; Isotopes; Label; Larva; Lead; Lung diseases; Mediating; Membrane Proteins; Methodology; Methods; Moths; Mutagenesis; NMR Spectroscopy; Neurotransmitters; Norepinephrine; Nuclear Magnetic Resonance; Nucleopolyhedrovirus; Obesity; Pharmaceutical Preparations; Plants; Preparation; Production; Proteins; Protocols documentation; Public Health; Recombinant Proteins; Research; Research Design; Resolution; Sampling; Signal Pathway; Source; Staging; Structure; System; Technology; X-Ray Crystallography; drug discovery; feeding; instrumentation; member; milligram; novel therapeutics; programs; protein structure; quantum; receptor; structural biology; tool

DESCRIPTION (provided by applicant): The goal of this proposal is to enable the application of Nuclear Magnetic Resonance (NMR) spectroscopy to investigate G protein coupled receptor (GPCR) structure and facilitate drug discovery. G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are the largest group of targets for novel therapeutics. The focus of research in my lab has been to characterize the structure and mechanism of activation of GPCRs using the ¿2 adrenergic receptor (¿2AR) as a model system. The ¿22AR is one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine, and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. During the past several years my lab has made significant progress towards obtaining a high-resolution structure of the ¿2AR by X-ray crystallography, as well as characterizing the structural changes associated with agonist activation. Biophysical studies on purified ¿2AR protein provide evidence that GPCRs are conformationally complex molecules. This conformational complexity contributes to the challenges of drug discovery: in identifying lead compounds, and in developing leads into selective, effective and safe drugs. Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool that has the potential to provide high-resolution structural information about receptor-drug interactions and about the dynamic aspects of GPCR structure. The goal of this R21 proposal is to develop expression technology to make NMR analysis of GPCRs and other membrane proteins economically tractable, and to enable the routine use of NMR for GPCR structural biology and drug discovery programs. We propose to achieve this goal by developing Trichoplusia ni (T. ni) insect larvae as an expression and isotope labeling system for GPCRs. T. ni are a natural host for baculovirus and have been used to express recombinant proteins in milligram quantities. Moreover, T. ni eat a variety of plant material and can be fed inexpensive sources of 13C and 15N: the isotopes needed for high-resolution NMR. Specific Aims include: AIM 1 - Develop economical Trichoplusia ni diet for 13C and 15N isotope labeling of recombinant proteins expressed in baculovirus infected larvae. AIM 2 - Develop efficient protocols for large-scale baculovirus-mediated expression of the ¿2AR in Trichoplusia ni larvae. AIM 3 - Optimize purification of functional ¿2AR from Trichoplusia ni larvae. PUBLIC HEALTH RELEVANCE GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug discovery. The proposed studies will develop the technology to make labeling of GPCR protein with 13C and 15N economically tractable and broadly applicable, and will enable the routine use of Nuclear Magnetic Resonance spectroscopy for GPCR structural biology and drug discovery programs. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

描述（由申请人提供）：本提案的目标是使核磁共振（NMR）光谱学能够应用于研究G蛋白偶联受体（GPCR）结构并促进药物发现。G蛋白偶联受体（GPCR）构成人类基因组中最大的膜蛋白家族，并且是新型治疗剂的最大组靶点。在我的实验室的研究重点一直是使用<$2肾上腺素能受体（<$2AR）作为模型系统来表征GPCR的结构和激活机制。22 AR是GPCR家族中最广泛表征的成员之一。它对儿茶酚胺类神经递质肾上腺素、去甲肾上腺素和多巴胺有反应，并且从广泛的诱变研究中了解了许多关于其激动剂结合和G蛋白偶联结构域的信息。在过去的几年里，我的实验室在通过X射线晶体学获得<$2AR的高分辨率结构以及表征与激动剂激活相关的结构变化方面取得了重大进展。对纯化的<$2AR蛋白的生物物理学研究提供了GPCR是构象复杂分子的证据。这种构象的复杂性有助于药物发现的挑战：在识别先导化合物，并在开发铅成选择性，有效和安全的药物。核磁共振（NMR）光谱是一种多功能的工具，有可能提供有关受体-药物相互作用和GPCR结构动态方面的高分辨率结构信息。该R21提案的目标是开发表达技术，使GPCR和其他膜蛋白的NMR分析经济上易于处理，并使NMR在GPCR结构生物学和药物发现计划中的常规使用成为可能。我们建议通过开发粉纹夜蛾Trichoplusia ni（T. ni）昆虫幼虫作为GPCR的表达和同位素标记系统。t. NI是杆状病毒的天然宿主，并已用于以毫克数量表达重组蛋白。此外，T.镍可以吃各种植物材料，并可以获得廉价的13 C和15 N来源：高分辨率NMR所需的同位素。具体目标包括：目的1-研制经济的粉纹夜蛾饲料，用于13 C和15 N同位素标记杆状病毒感染幼虫表达的重组蛋白。目的2-开发大规模杆状病毒介导的在粉纹夜蛾幼虫中表达Δ 2AR的有效方案。目的3-优化粉纹夜蛾幼虫中功能性<$2AR的纯化。GPCR代表了人类基因组中最大的膜蛋白家族，也是最大的药物发现靶点。拟议的研究将开发技术，使13 C和15 N标记的GPCR蛋白质经济上易于处理和广泛适用，并将使常规使用的核磁共振光谱的GPCR结构生物学和药物发现计划。作用于GPCR的药物可以对广泛的疾病产生影响，包括：心血管疾病、肺部疾病、炎症、糖尿病和肥胖症、行为障碍和阿尔茨海默病。