Abstract
Salt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulation is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depends on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible lysine-carboxylate ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is overestimated, to different extents, in simulations of GB1 using seven out of eight commonly used combinations of fixed charge force fields and water models. We also find that the Amber ff15ipq force field gives rise to weaker salt bridges in good agreement with the NMR experiments. We conclude that many force fields appear to overstabilize these ionic interactions, and that further work may be needed to refine our ability to model quantitatively the stability of salt bridges through simulations. We also suggest that comparisons between NMR experiments and simulations will play a crucial role in furthering our understanding of this important interaction.