Biochemical Characterization of Cell-free Synthesized Human β1 Adrenergic Receptor Cotranslationally Inserted into Nanodiscs. Issue 16 (30th August 2022)

• Record Type: Journal Article
• Authors: Köck, Zoe; Ermel, Utz; Martin, Janosch; Morgner, Nina; Frangakis, Achilleas S.; Dötsch, Volker; Hilger, Daniel; Bernhard, Frank
• Is Part Of: Journal of molecular biology. Volume 434:Issue 16(2022)
• Publication Date: 2022-08-30
• Subjects: G-protein coupled receptors -- cell-free expression -- nanodiscs -- G protein coupling -- single particle analysis; βME β-Mercaptoethanol -- CF Cell-free -- CHS Cholesteryl hemisuccinate -- CL Cardiolipin -- DDM Dodecylmaltoside -- DEPG 1, 2-Dielaidoyl-sn-glycero-3-phospho-(1′-rac-glycerol) -- DMPC 1, 2-Dimyristol-sn-glycero-3-phosphocholine -- DMPG 1, 2-Dimyristol-sn-glycero-3-phospho-(1′-rac-glycerol) -- DOPC 1, 2-Dioleoyl-sn-glycero-3-phosphocholine -- DOPE 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine -- DOPG 1, 2-Dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) -- E. coli TL E. coli total lipid extract -- GDP Guanosine diphosphate -- GFP Green fluorescent protein -- GPCR G protein coupled receptor -- GTP Guanosine triphosphate -- Hβ1AR human β1 adrenergic receptor -- HtsFLβ1AR human thermostabilized full-length β1 adrenergic receptor -- HtsΔβ1AR human thermostabilized truncated β1 adrenergic receptor -- ICL3 Intracellular loop 3 -- IMAC Immobilized metal affinity chromatography -- LILBID Laser induced liquid bead ion desorption -- Mini-G Minimal G protein -- MS Mass spectroscopy -- MSP Membrane scaffold protein -- MWCO Molecular weight cut-off -- NDs Nanodiscs -- RM Reaction mixture -- SEC Size exclusion chromatography; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2022.167687
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: Optimized cell-free expression protocol to cotranslationally insert full-length human β1 AR into preformed nanodiscs. Pharmacological profiling of full-length and truncated human β1 AR derivatives in defined membrane environments. Biochemical in vitro analysis of the G/R389 polymorphism. Detergent-free production and negative stain analysis of full-length human β1 AR samples coupled to Gs heterotrimer. 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-Gαs protein as well as to purified Gs heterotrimer could be demonstrated and homogeneity of purified GPCR/Gs protein complexes in nanodiscs was demonstrated by negative stain single particle analysis.