Uncovering pH-Dependent Transient States of Proteins with Buried Ionizable Residues

[Image: see text] The role of pH in regulating biological activity is ubiquitous, and understanding pH-mediated activity has traditionally relied on analyzing static biomolecular structures of highly populated ground states solved near physiological pH. However, recent advances have shown the increasing importance of transiently populated states, the characterization of which is extremely challenging but made plausible with the development of techniques such as relaxation dispersion NMR spectroscopy. To unlock the pH dependence of these transient states with atomistic-level details, we applied the recently developed explicit solvent constant pH molecular dynamics (CPHMD(MSλD)) framework to a series of staphylococcal nuclease (SNase) mutants with buried ionizable residues and probed their dynamics in different pH environments. Among our key findings is the existence of open states in all SNase mutants containing “buried” residues with highly shifted pK(a)’s, where local solvation around the protonation site was observed. The calculated pK(a) demonstrated good agreement with experimental pK(a)’s, with a low average unsigned error of 1.3 pK(a) units and correlation coefficient R(2) = 0.78. Sampling both open and closed states in their respective pH range, where they are expected to be dominant, was necessary to reproduce experimental pK(a)’s, and in the most extreme examples of pK(a) shifts measured, it can be interpreted that the open-state structures are transient at physiological pH, contributing a small population of 1–2%. This suggests that buried ionizable residues can trigger conformational fluctuations that may be observed as transient-state structures at physiological pH. Furthermore, the coupled relationship of both open and closed states and their role in recapitulating macroscopic experimental observables suggest that structural analysis of buried residues may benefit from looking at structural pairs, as opposed to the conventional approach of looking at a single static ground-state conformation.