Predicting pKa values with continuous constant pH molecular dynamics

Knowledge of pK(a) values is important for understanding structure and function relationships in proteins. Over the past two decades, theoretical methods for pK(a) calculations have been mainly based on macroscopic models, in which the protein is considered as a low-dielectric cavity embedded in a high-dielectric continuum. In recent years, constant pH molecular dynamics methods have been developed based on a microscopic description of the protein. We describe here the methodology of continuous constant pH molecular dynamics (CPHMD), which has emerged as one of the most robust and accurate tools for predicting protein pK(a)s and for the study of pH-modulated conformational dynamics. We illustrate the utility of CPHMD by the calculation of pK(a)s for surface residues in ribonuclease A, buried residues in staphylococcal Nuclease, and titratable groups in the intrinsically flexible protein α-lactalbumin. We will compare the CPHMD results with experimental data as well as calculations from PB-based and empirical methods. These examples demonstrate the accuracy and robustness of the CPHMD method and its ability to capture the correlation between ionization equilibria and conformational dynamics as well as the local dielectric response to structural rearrangement. Finally, we discuss future improvement of the CPHMD method.