MUTANT G-PROTEIN COUPLED RECEPTORS AND METHODS FOR SELECTING THEM
The invention relates to mutant G-protein coupled receptors with increased conformational stability, and methods of use thereof. In some aspects, polynucleotides encoding the mutant G-protein coupled receptors are provided. In some aspects, host cells comprising the polynucleotides are provided. In some aspects, the invention relates to crystallized forms of the mutant G-protein coupled receptors, and methods of preparing the same.
1 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability compared to its parent GPCR, which mutant GPCR has an extended lifetime of a particular conformation relative to the same conformation of its parent GPCR under denaturing conditions, the extended lifetime being manifest by retention of ligand binding ability.
2 . A mutant GPCR according to claim 1 , wherein the mutant GPCR has an extended lifetime under conditions selected from any one or more of heat, a detergent, a chaotropic agent and an extreme of pH.
3 . A mutant GPCR according to claim 2 , wherein the mutant GPCR has increased conformational thermostability, optionally which is at least 1° C. more stable than its parent.
4 . A mutant GPCR according to claim 1 , with increased conformational stability of an agonist or antagonist conformation.
5 . A mutant GPCR according to claim 1 , wherein the mutant GPCR is a Class 1 GPCR.
6 . A mutant GPCR according to claim 1 , wherein the mutant GPCR is a Class 2 GPCR.
7 . A mutant GPCR according to claim 1 , wherein the mutant GPCR is a Class 3 GPCR.
8 . A polynucleotide encoding a mutant GPCR according to claim 1 .
9 . A host cell comprising a polynucleotide according to claim 8 .
10 . A mutant GPCR according to claim 1 , which is in a solubilized form.
11 . A mutant GPCR according to claim 1 , which is substantially free of other proteins.
12 . A mutant GPCR according to claim 1 , which is immobilized to a solid support.
13 . A mutant GPCR according to claim 1 , wherein the mutant GPCR is not a human cannabinoid receptor.
14 . A polynucleotide according to claim 8 , wherein the mutant GPCR is not a human cannabinoid receptor.
15 . A host cell according to claim 9 , wherein the mutant GPCR is not a human cannabinoid receptor.
16 . A crystallized form of a mutant GPCR according to claim 1 .
17 . A method comprising crystallizing a mutant GPCR of claim 1 .
18 . A method comprising
obtaining a mutant GPCR of claim 1 , and
screening for a ligand that binds to the mutant GPCR.
19 . The method of claim 18 , wherein the mutant GPCR is coupled to a solid surface of a biosensor chip.
20 . A method comprising
obtaining a mutant GPCR of claim 1 , and
developing an assay involving binding of a ligand to the mutant GPCR.