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NameBeining Jin
Organization or InstitutionUniversity of Florida
TopicPhysical Chemistry
Title

Investigating the Conformational Dynamics of the Human A2A Adenosine Receptor in Lipid Vesicles by 19F MAS Solid-State NMR

Author(s)

Beining Jin, Arka Prabha Ray and Matthew T. Eddy

Author Institution(s)

Department of Chemistry, University of Florida

Abstract

With more than 800 distinct proteins encoded by the human genome, G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins and top drug targets in academic and industrial labs. Insights into GPCR structural plasticity have largely come from NMR spectroscopic studies. To date, however, the vast majority of GPCR NMR studies have utilized preparations of GPCRs in aqueous solutions containing membrane mimetics such as detergent micelles or lipid nanodiscs. While useful, such membrane mimetics differ in their bulk physical properties from other membrane systems such as lipid vesicles, which can better represent conditions present in the cellular environment.
We present optimized protocols for preparing samples of the human A2A adenosine receptor (A2AAR), a representative class A GPCR, in unilamellar vesicles for 19F magic angle spinning (MAS) solid-state NMR spectroscopy. Utilizing multiple functional and biophysical assays, we confirmed that vesicles containing 19F-labeled A2AAR bound ligands with native affinities, and we quantified the density and relative orientation of receptors in lipid vesicles. By optimizing spectral dispersion and resolution as a function of MAS frequency and 1H decoupling strength, we obtained 19F MAS SSNMR data of similar spectral resolution and dispersion as preparations with lipid nanodiscs in an aqueous solution. The similar resolution and dispersion facilitated direct comparisons of the conformational equilibria of A2AAR between lipid nanodiscs and unilamellar vesicles. This provided new insights into the potential role of bulk membrane properties, such as lipid curvature, in modulating the conformational landscape of A2AAR.