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

描述（由申请人提供）：本提案的目标是应用核磁共振（NMR）波谱来研究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）是人类基因组中最大的膜蛋白家族，也是新疗法的最大靶点。我的实验室的研究重点是用¿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。¿22AR是GPCR家族中最广泛表征的成员之一。它对儿茶酚胺神经递质肾上腺素、去甲肾上腺素和多巴胺有反应，并且从广泛的诱变研究中对其激动剂结合和G蛋白偶联域有很多了解。在过去的几年中，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。纯化的¿2AR蛋白的生物物理学研究提供了证据，证明gpcr是构象复杂的分子。这种构象复杂性增加了药物发现的挑战：在识别先导化合物方面，以及在将先导物开发成选择性、有效和安全的药物方面。核磁共振（NMR）光谱是一种多功能工具，具有提供受体-药物相互作用和GPCR结构动态方面的高分辨率结构信息的潜力。本R21提案的目标是开发表达技术，使GPCR和其他膜蛋白的核磁共振分析经济容易处理，并使核磁共振常规应用于GPCR结构生物学和药物发现项目。为了实现这一目标，我们建议开发一种用于gpcr表达和同位素标记的昆虫幼虫。T. ni是杆状病毒的天然宿主，已被用于表达毫克级的重组蛋白。此外，T. ni吃各种植物材料，并且可以获得廉价的13C和15N来源：高分辨率核磁共振所需的同位素。具体目标包括：AIM 1 -开发经济型ni毛梳虫饲料，用于杆状病毒感染幼虫中表达的重组蛋白的13C和15N同位素标记。AIM 2 -开发杆状病毒介导的在ni毛虫幼虫中大规模表达¿2AR的有效方案。目的3 -优化纯化ni毛虫幼虫的功能性¿2AR。gpcr代表了人类基因组中最大的膜蛋白家族，也是药物发现的最大靶点。本研究将发展用13C和15N标记GPCR蛋白的技术，使其在经济上易于处理和广泛应用，并将使核磁共振波谱技术在GPCR结构生物学和药物发现项目中的常规应用成为可能。作用于gpcr的药物可对多种疾病产生影响，包括：心血管疾病、肺病、炎症、糖尿病和肥胖症、行为障碍和阿尔茨海默病。

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.