Conferring thermostability to mesophilic proteins through optimized electrostatic surfaces.

Recently, there have been several experimental reports of proteins displaying appreciable stability gains through mutation of one or two amino acid residues. Here, we employ a simple theoretical model to quickly screen mutant structures for increased thermostability through optimization of the protein's electrostatic surface. Our results are able to reproduce the experimental observation that elimination of like-charge repulsions and creation of opposite-charge attractions on the protein surface is an efficient method to confer thermostability to a mesophilic protein. Using Poisson-Boltzmann electrostatics, we calculate relative protein stabilities for the exhaustive surface mutagenesis of the cold shock, RNase T1, and CheY proteins. Comparison with 25 experimentally characterized cold shock protein mutants reveals an average correlation of 0.86. The model is also quantitatively accurate when reproducing the experimental D49A and D49H mutant stabilities of RNase T1. This work represents the first comprehensive in silico screening of mutant candidates likely to confer thermostability to mesophilic proteins through optimization of surface electrostatics. Systematic single mutant, followed by double mutant, screening yields a limited number of mutant structures displaying significant stability gains suitable for subsequent experimental characterization.