A pH-dependent computational approach to the effect of mutations on protein stability

This article describes a novel software implementation for high-throughput scanning mutagenesis with a focus on protein stability. The approach combines molecular mechanics calculations with calculations of protein ionization and a Gaussian-chain model of electrostatic interactions in unfolded state. Comprehensive testing demonstrates a state-of-the-art accuracy for predicted free energy differences on single, double, and triple mutations with a correlation coefficient R above 0.7, which takes about 1.5 min per mutation on a single CPU. Unlike most of existing in silico methods for fast mutagenesis, the stability changes are reported as a continuous function of solution pH for wide pH intervals. We also propose a novel in silico strategy for searching stabilized protein variants that is based on combinatorial scanning mutagenesis using representative amino acid types. Our in silico predictions are in excellent agreement with the hyper-stabilized variants of mesophilic cold shock protein found using the Proside method of direct evolution. © 2016 Wiley Periodicals, Inc.