| Abstract
| - The calculation of pKa values for ionizable sites in proteins has been traditionally based onnumerical solutions of the Poisson-Boltzmann equation carried out using a high-resolution protein structure.In this paper, we present a method based on continuous constant pH molecular dynamics (CPHMD)simulations, which allows the first-principles description of protein ionization equilibria. Our methodutilizes an improved generalized Born implicit solvent model with an approximate Debye-Hückel screeningfunction to account for salt effects and the replica-exchange (REX) protocol for enhanced conformationaland protonation state sampling. The accuracy and robustness of the present method are demonstrated by1 ns REX-CPHMD titration simulations of 10 proteins, which exhibit anomalously large pKa shifts forthe carboxylate and histidine side chains. The experimental pKa values of these proteins are reliablyreproduced with a root-mean-square error ranging from 0.6 unit for proteins containing few buried ionizableside chains to 1.0 unit or slightly higher for proteins containing ionizable side chains deeply buried in thecore and experiencing strong charge−charge interactions. This unprecedented level of agreement withexperimental benchmarks for the de novo calculation of pKa values suggests that the CPHMD method ismaturing into a practical tool for the quantitative prediction of protein ionization equilibria, and this, inturn, opens a door to atomistic simulations of a wide variety of pH-coupled conformational phenomenain biological macromolecules such as protein folding or misfolding, aggregation, ligand binding, membraneinteraction, and catalysis.
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