We developed a Rosetta-based Monte Carlo method to calculate the pK a values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pKa values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pKa shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pKa calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pKa predictions for proximal catalytic residues. The method also captures large pKa shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK a values that will be useful when rapid estimation is essential, such as in docking, design, and folding.
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