Microscopic mechanisms that govern the titration response and pKa values of buried residues in staphylococcal nuclease mutants

To probe the microscopic mechanisms that govern the titration behavior of buried ionizable groups, microsecond explicit solvent molecular dynamics simulations are carried out for several mutants of Staphylococcal nuclease using both fixed charge and polarizable force fields. While the ionization of Asp 66, Glu 66, and Lys 125 lead to enhanced structural fluctuations and partial unfolding of adjacent α‐helical regions, the ionization of Lys 25 causes local unfolding of adjacent β sheets. Using the sampled conformational ensembles, good agreement with experimental pK_a values is obtained with Poisson–Boltzmann calculations using a protein dielectric constant of 2–4 for V66D/E; slightly larger dielectric constants are needed for Lys mutants especially L25K, suggesting that structural responses beyond microseconds are involved in ionization of Lys 25. Overall, the set of unbiased simulations provides insights into the spatial and temporal scales of protein and solvent motions that dictate the diverse titration behaviors of buried protein residues. Proteins 2017; 85:268–281. © 2016 Wiley Periodicals, Inc.