title: Purification of Stabilized GPCRs for Structural and Biophysical Analyses.
abstract: G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor toward a specific physiologically relevant conformational state. The resultant stabilized receptor (known as a StaR) can be purified in multiple-milligram quantities, whilst retaining correct folding, thus enabling the generation of reagents suitable for a broad range of structural and biophysical studies. Example protocols for the purification of StaR proteins for analysis, ligand screening with the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), surface plasmon resonance (SPR), and crystallization for structural studies are presented.
authors.affiliation: Heptares Therapeutics, Biopark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: James C
authors.initials: JC
authors.lastname: Errey
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Doré
authors.forename: Andrei
authors.initials: A
authors.lastname: Zhukov
authors.forename: Fiona H
authors.initials: FH
authors.lastname: Marshall
authors.forename: Robert M
authors.initials: RM
authors.lastname: Cooke
mesh_terms: D055592:Biophysical Phenomena; D005591:Chemical Fractionation / Q000379:methods*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D055767:Immobilized Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008024:Ligands; D008301:Maleimides / Q000737:chemistry; D055550:Protein Stability; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D008301:Maleimides; D043705:Receptor, Adenosine A2A; D018094:Receptors, Metabotropic Glutamate; D014970:Xanthines; C043592:maleimide; C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine