The role of pH-dependent protonation equilibrium in modulating RNA dynamics and function is one of the key unanswered questions in RNA biology. Molecular dynamics (MD) simulations can provide insight into the mechanistic roles of protonated nucleotides, but it is only capable of modeling fixed protonation states and requires prior knowledge of the key residue's protonation state. Recently, we developed a framework for constant pH molecular dynamics simulations (CPHMDMSλD) of nucleic acids, where the nucleotides' protonation states are modeled as dynamic variables that are coupled to the structural dynamics of the RNA. In the present study, we demonstrate the application of CPHMDMSλD to the lead-dependent ribozyme; establishing the validity of this approach for modeling complex RNA structures. We show that CPHMDMSλD accurately predicts the direction of the pKa shifts and reproduces experimentally-measured microscopic pKa values with an average unsigned error of 1.3 pKa units. The effects of coupled titration states in RNA structures are modeled, and the importance of conformation sampling is highlighted. The general accuracy of CPHMDMSλD simulations in reproducing pH-dependent observables reported in this work demonstrates that constant pH simulations provides a powerful tool to investigate pH-dependent processes in nucleic acids.
Keywords: CPHMD; constant pH molecular dynamics; pKa; λ-dynamics.