Constant-pH molecular dynamics study of protonation-structure relationship in a heptapeptide derived from ovomucoid third domain

Molecular dynamics (MD) simulations with implicit solvent and variable protonation states for titratable residues at constant pH are performed for a short peptide derived from ovomucoid third domain (OMTKY3), acetyl-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-amide (residues 26–32 of OMTKY3). Nuclear magnetic resonance (NMR) measurements indicate that the ${\mathrm{pK}}_a$ for Asp is 3.6. However, if the charge on Lys is neutralized by acetylation, then the $p{\mathrm{K}}_a$ for Asp is 4.0. These $p{\mathrm{K}}_a\mathrm{’}\mathrm{s},$ and therefore the Asp-Lys interaction, are insensitive to changes in ionic strength. The constant-pH MD simulations for both variants of the heptapeptide yield Asp $p{\mathrm{K}}_a$ values that are 0.6–0.9 pH units greater than experimental values, but the difference between the variants that is observed in the NMR experiments is reproduced much better. Moreover, the simulations suggest that Asp-Lys interactions do not dominate the behavior of this heptapeptide, even for normal Lys residue where there is a possibility of forming a salt bridge between negatively charged Asp and positively charged Lys. This is consistent with the experimentally observed independence of Asp $p{\mathrm{K}}_a$ values with respect to ionic strength. Another important result of the simulations with variable protonation states is that they lead to ensembles of the heptapeptide structures that are different from those derived from simulations with fixed protonation states. It should be stressed that these results are for structures generated entirely by computer simulations without any restrictions imposed by experimental data.