The key to predicting the stability of protein mutants lies in an accurate description and proper configurational sampling of the folded and denatured states.
The contribution of particular hydrogen bonds to the stability of a protein fold can be investigated experimentally as well as computationally by the construction of protein mutants which lack particular hydrogen-bond donors or acceptors with a subsequent determination of their structural stability. However, the comparison of experimental data with computational results is not straightforward. One of the difficulties is related to the representation of the unfolded state conformation. A series of molecular dynamics simulations of the 34-residue WW domain of protein Pin1 and 20 amide-to-ester mutants started from the X-ray crystal structure and the NMR solution structure are analysed in terms of backbone-backbone hydrogen bonding and differences in free enthalpies of folding in order to provide a structural interpretation of the experimental data available. The contribution of the different β-sheet hydrogen bonds to the relative stability of the mutants with respect to wild type cannot be directly inferred from experimental thermal denaturation temperatures or free enthalpies of chaotrope denaturation for the different mutants, because some β-sheet hydrogen bonds show sizeable variation in occurrence between the different mutants. A proper representation of unfolded state conformations appears to be essential for an adequate description of relative stabilities of protein mutants. The simulations may be used to link the structural Boltzmann ensembles to relative free enthalpies of folding between mutants and wild-type protein and show that unfolded conformations have to be treated with a sufficient level of detail in free energy calculations of protein stability. This article is part of a Special Issue entitled Recent developments of molecular dynamics.