Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pKa prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pKa predictions but also inform about the physical origins of pKa shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pKa prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pKa values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pKa's with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pKa values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pKa predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy. Proteins 2011. © 2011 Wiley-Liss, Inc.