Biochemical Characterization of Cell-free Synthesized Human β1 Adrenergic Receptor Cotranslationally Inserted into Nanodiscs

Abstract

Cell-free expression enables direct cotranslational insertion of G protein coupled receptors (GPCRs) and other membrane proteins into the defined membrane environments of nanodiscs. This technique avoids GPCR contacts with detergents and allows rapid identification of lipid effects on GPCR function as well as fast screening of receptor derivatives. Critical steps of conventional GPCR preparation from cellular membranes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human β₁-adrenergic receptor and of a truncated derivative into preformed nanodisc membranes. Their biochemical characterization revealed significant differences in lipid requirements, dimer formation and ligand binding activity. The truncated receptor showed a higher affinity to most tested ligands, in particular in presence of choline-containing lipids. However, introducing the naturally occurring G389R polymorphism in the full-length receptor resulted into an increased affinity to the antagonists alprenolol and carvedilol. Receptor quality was generally improved by coexpression with the agonist isoproterenol and the percentage of the ligand binding active fraction was twofold increased. Specific coupling of full-length and truncated human receptors in nanodisc membranes to Mini-Gα_s protein as well as to purified G_s heterotrimer could be demonstrated and homogeneity of purified GPCR/G_s protein complexes in nanodiscs was demonstrated by negative stain single particle analysis.

Keywords: G protein coupling; G-protein coupled receptors; cell-free expression; nanodiscs; single particle analysis.