Energetic fitness of histidine protonation states in PDB structures

The energetic fitness of histidine in each of its three protonation states has been investigated for NMR-determined protein structures by using molecular mechanics calculations. The protein structures have been taken from the Protein Data Bank (PDB). For the proteins in the database, we generated all isomers, considering all combinations of protonation forms of each histidine. The energy of each isomer has been determined by conjugate gradient minimization using a well-established all-atom force field. We find that, in general, the isomer reported in the PDB is not the most stable isomer. The statistical distribution of isomer energies minus that of the PDB isomer behaves as though the sequence of the histidine forms reported in the PDB was chosen at random. We also show that our molecular mechanics method is a valid approach to predicting the protonation state of histidines buried in the protein core.