Prevalence and mechanism of synergistic carboxylate-cation-water interactions in halophilic proteins.

The cytoplasmic proteins of some halophilic organisms remain stable and functional at multimolar concentrations of KCl, i.e., under conditions that most mesophilic proteins cannot withstand. Their stability arises from their unusual amino acid composition. The most dramatic difference between halophilic and mesophilic proteins is that the former are rich in acidic amino acids. It has been proposed that one of the evolutionary driving forces for this difference is the occurrence of synergistic interactions between multiple acidic amino acids at the surface of the protein, the potassium cations in solution, and water. We investigate this possibility with molecular dynamics simulations, using high-quality force fields for the protein-water, protein-ion, and ion-ion interactions. We create a rigorous thermodynamic definition of interactions between acidic amino acids on proteins that can be used to distinguish between synergistic, noninteracting and interfering interactions. Our results demonstrate that synergistic interactions between neighboring acidic amino acids in halophilic proteins are frequent at multimolar KCl concentration. Synergistic interactions have an electrostatic origin, and are associated with stronger water-to-carboxylate hydrogen bonds than for acidic amino acids without synergistic interactions. Synergistic interactions are not observed in minimal systems of carboxylates, indicating that the protein environment is critical for their emergence. Our results demonstrate that synergistic interactions are neither associated with rigid amino acid orientations nor with highly structured and slow moving water networks, as had been originally proposed. Moreover, synergistic interactions can also be found in unfolded protein conformations. However, because these conformations are only a small subset of the unfolded state ensemble, synergistic interactions should contribute to the net stabilization of the folded state.