Given the discussions in the literature pertaining to ion overbinding and the role of a solvent model on protein solvation 135 , we began by assessing the role of environmental conditions. Specifically, we probed K + (rather than Na + ) counterions, NBFIX parameters 133 , Åqvist 136 (rather than Joung/Cheatham 137 ) ion parameters, and TIP4P-D water 138 (rather than TIP3P). Using these variants, the pK a values of lysines from a 13 residue dataset (i.e., hen egg-white lysozyme (HEWL) and calbindin 9k) were computed. No significant improvement in the estimates was observed (Figure ).