BPS2026 – Conformational dynamics and protonation in membrane proteins: Insights from CpHMD simulations

Recent studies highlight how residue conformation significantly impacts protonation curves, deviating from the Henderson-Hasselbalch model. Constant pH molecular dynamics (CpHMD) simulations of membrane proteins like OmpF and others reveal that side-chain fluctuations create heterogeneous environments, resulting in non-ideal titration behavior. For example, titration curves of key residues such as D97 and E117 in OmpF exhibit Hill coefficients below 1 (0.3–0.4), suggesting an apparent negative cooperativity possibly resulting from microstate heterogeneity rather than from simple electrostatic interactions. Additionally, applied voltage modulates local pH gradients across membrane-spanning regions, directly affecting apparent pKa values. In these channels, this electrokinetic coupling can shift protonation states of key residues by altering the electrostatic potential landscape. Such effects are particularly pronounced at transmembrane interfaces, where voltage-driven ion accumulation creates highly localized pH microenvironments. These dynamic interactions could explain discrepancies between static pKa predictions and functional data under physiological conditions. Earlier experimental evidence shows that in the OmpF bacterial porin, unlike what occurs in monovalent cation salts, its selectivity is practically insensitive to pH. Anionic selectivity is maintained throughout the acidic pH range. Electrostatic analyses suggested that Mg 2+ binding can affect the pKa values of key ionizable groups, which differ significantly from those of the isolated groups in solution. Building on these insights, we now turn to constant pH molecular dynamics (CpHMD) simulations, which provide a more direct and dynamic framework for characterizing how divalent cations modulate residue protonation and channel selectivity under physiologically relevant conditions.