LAUSR

Protonation states of titratable amino acids frequently play a key role in many biomolecular processes. Knowledge of protonatable residue charges at a given pH is essential for a correct understanding of protein catalysis, inter- and intra- molecular interactions, substrate binding, protein dynamics for instance. However, acquiring experimental values for individual amino acid protonation states of complex systems is not straightforward; therefore, several in silico approaches have been developed to tackle this issue. In this work, we assess the accuracy of our previously developed constant pH MD approach by comparing our theoretically obtained pKa values for titratable residues, with experimental values from an equivalent NMR study. We selected a set of four pentapeptides, of adequate small size to ensure comprehensive sampling, but concurrently, due to their charge composition, posing a challenge for protonation state calculation. The comparison of the pKa values shows good agreement of the experimental and the theoretical approach with a largest difference of 0.25 pKa units. Further, the corresponding titration curves are in fair agreement although the shift of the Hill coefficient from value of 1 was not always reproduced in simulations. The phase space overlap in Cartesian space between trajectories generated in constant pH and standard MD simulations is fare and suggests that our constant pH MD approach reasonably well preserves the dynamics of the system, allowing dynamic protonation MD simulations without introducing structural artefacts.