Mutant G-protein coupled receptors and methods for selecting them

A method for selecting a G-protein coupled receptor (GPCR) with increased conformational thermostability in a particular conformation compared to its parent GPCR in the same particular conformation, which particular conformation is selected from an agonist conformation and an antagonist conformation, the method comprising

• (a) preparing one or more mutants of a parent GPCR, wherein the one or more mutants each differs from its parent GPCR by one or more point mutations and wherein the one or more mutants and the parent GPCR are provided in a solubilised form obtained by solubilising the one or more mutants and the parent GPCR from a membrane using a detergent;
• (b) contacting the one or more mutants and the parent GPCR with a ligand of a particular class, wherein:
• (i) an agonist ligand is used to select for one or more mutants with increased conformational stability in the agonist conformation by binding to the one or more mutants;
• and
• (ii) an antagonist ligand is used to select for one or more mutants with increased conformational stability in the antagonist conformation by binding to the one or more mutants;
• (c) exposing to heat the one or more mutants and the parent GPCR, wherein the one or more mutants and the parent GPCR are bound to the ligand;
• (d) measuring the stability of binding of the one or more mutants and the parent GPCR to the ligand by measuring the ability of the one or more mutants and the parent GPCR to retain ligand binding capacity following exposure to heat;
• and
• (e) selecting the one or more mutants that exhibit increased stability of binding to the ligand following exposure to heat.