Can the protonation state of histidine residues be determined from molecular dynamics simulations?

Histidine (His) residues in proteins can attain three different protonation states at normal pH. This constitutes a prominent problem when adding protons to a protein crystal structure, e.g. in order to perform molecular simulations. Typically, the His protonation is deduced from the hydrogen-bond pattern in crystal structures. Here, we study whether it is possible to detect erroneous His protonation state by analysing short molecular dynamics (MD) trajectories. We systematically vary the His protonation state and measure the root-mean-squared deviation (RMSD) of the His residues and nearby residues relative to the starting structure, as well as the distribution of the dihedral angle that determines the rotation of the His side chain. We study three proteins, hisactophilin with 31 solvent-exposed His residues, galectin-3, for which an experimental assignment is available for two of the His residues, and trypsin, for which the hydrogen-bond analysis is quite conclusive. The results show that improper protonation states have larger RMSD values and larger widths of the dihedral distribution, compared to the correct protonation states. Unfortunately, the variation among different His residues in the same and different proteins is so large that it is hard to define unambiguous thresholds between proper and improper protonation states. Therefore, simulations of all three protonation states are needed for conclusive results. For trypsin, we could obtain a conclusive assignment for all three His residues, which was better than the simple hydrogen-bond analysis. For galectin-3, the MD trajectories confirmed the results of hydrogen-bond analysis and experiments. They also gave additional, more uncertain information for some of the residues. However, for the solvent-exposed His residues in hisactophilin, no unambiguous conclusions regarding the protonation states could be reached. On the other hand, this indicates that protein structures are quite insensitive to the protonation state of the His residues, besides those that involve direct hydrogen bonds to the His side chain. (C) 2012 Elsevier B.V. All rights reserved.