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

Stanford University, Stanford CA

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Abstract

[unreadable] 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 [unreadable]2 adrenergic receptor ([unreadable]2AR) as a model system. The [unreadable]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 [unreadable]2AR by X-ray crystallography, as well as characterizing the structural changes associated with agonist activation. Biophysical studies on purified [unreadable]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 [unreadable]2AR in Trichoplusia ni larvae. AIM 3 - Optimize purification of functional [unreadable]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. [unreadable] [unreadable] [unreadable]