Nonlinear impact of electrolyte solutions on protein dynamics.

Halophilic organisms have adapted to multi-molar salt concentrations, their cytoplasmic proteins functioning despite stronger attraction between hydrophobic groups. These proteins, of interest in biotechnology because of decreasing fresh-water resources, have excess acidic amino acids.   It has been suggested that conformational fluctuations -- critical for protein function -- decrease in the presence of a stronger hydrophobic effect, and that an acidic proteome would counteract this decrease. However, our understanding of the salt- and acidic amino acid dependency of enzymatic activity is limited. Here, using solution NMR relaxation and molecular dynamics simulations for in total 14 proteins, we show that salt concentration has a limited and moreover non-monotonic impact on protein dynamics. The results speak against the conformational-fluctuations model, instead indicating that maintaining protein dynamics to ensure protein function is not an evolutionary driving force behind the acidic proteome of halophilic proteins.

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.

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids.

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (bKCl = 0.15 mol/kg) and high (bKCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.