Overcoming Convergence Issues in Free-Energy Calculations of Amide-to-Ester Mutations in the Pin1-WW Domain.

Relative folding free energies for a series of amide-to-ester mutations in the Pin1-WW domain are calculated using molecular dynamics simulations. Special focus is given to the identification and elimination of a simulation-related bias which was observed in previous work (Eichenberger et al. Biochim. Biophys. Acta 2015, 1850, 983) by comparing simulation results obtained with two different starting structures. Subtle local variations in the protein starting structure may lead to substantial deviations in the calculated free-energy changes as a consequence of differences in the sampled ϕ/ψ-dihedral angle distributions of the mutated residue. It is found that the combination of alchemical transformation with Hamiltonian replica exchange for enhanced sampling reduces the starting structure dependence considerably. Compared to previous work, the improved sampling of both the folded and unfolded states also improves the agreement between simulation and experiment.