Biochemical Characterization of Cell-free Synthesized Human ?1 Adrenergic Receptor Cotranslationally Inserted into Nanodiscs

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 mem-branes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human 81-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. How-ever, 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-Gas protein as well as to purified Gs heterotrimer could be demonstrated and homogeneity of puri-fied GPCR/Gs protein complexes in nanodiscs was demonstrated by negative stain single particle analysis. (C) 2022 Elsevier Ltd. All rights reserved.

Automated summary

Cell-free expression allows direct cotranslational insertion of GPCRs and other membrane proteins into nanodisc membranes, avoiding detergent contacts and enabling rapid lipid effect analysis and receptor derivative screening. The technique demonstrated efficient insertion of full-length and truncated human α-adrenergic receptors into preformed nanodisc membranes, revealing significant differences in lipid requirements, dimer formation, and ligand binding activity. Introduction of a natural polymorphism enhanced receptor affinity to specific antagonists. The coexpression of receptors with agonists improved receptor quality and ligand binding activity. Specific coupling to Mini-Gas protein and purified Gs heterotrimer in nanodisc membranes was also achieved, demonstrating the homogeneity of purified GPCR/Gs protein complexes by negative stain single particle analysis.