Abstract
Buried ionizable groups in proteins often play important structural and functional roles. However, it is generally challenging to study the detailed molecular mechanisms solely based on experimental measurements. Free energy calculations using atomistic simulations, on the other hand, complement experimental studies and can provide high temporal and spatial resolution information that can lead to mechanistic insights. Nevertheless, it is also well recognized that sufficient sampling of such atomistic simulations can be challenging, considering that structural changes related to the buried charges may be very slow. In the present study, we describe a simple but effective enhanced sampling technique called replica exchange with charge tempering (REChgT) with a novel free energy method, multisite λ dynamics (MSλD), to study two systems containing buried charges, pK(a) prediction of a small molecule, orotate, in complex with the dihydroorotate dehydrogenase, and relative stability of a Glu-Lys pair buried in the hydrophobic core of two variants of Staphylococcal nuclease. Compared to the original MSλD simulations, the usage of REChgT dramatically increases sampling in both conformational and alchemical spaces, which directly translates into a significant reduction of wall time to converge the free energy calculations. This study highlights the importance of sufficient sampling toward developing improved free energy methods.