A Methodology for Creating Thermostabilized Mutants of GPCR by Combining Statistical Thermodynamics and Evolutionary Molecular Engineering

Abstract

The thermostabilization of G-protein coupled receptors (GPCRs), which are important drug targets, is a crucial challenge for structural analysis. We constructed a methodology for thermostabilizing a GPCR in the inactive state or the active state solely by multiple amino-acid mutations without the ligand binding. This method combines our recently developed theory based on statistical thermodynamics and the technique of site-directed saturation mutagenesis, which is frequently used in evolutionary molecular engineering. The methodology was illustrated for the serotonin 2A receptor and the adenosine A_2A receptor, and we successfully obtained stabilized multiple mutants.