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

Abstract

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 (b_KCl = 0.15 mol/kg) and high (b_KCl = 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.

Figure 1

Mean molal activity coefficient $\left({\gamma }_{s,\pm }^{\left(b\right)}\right)$ of potassium acetate as a function of the molality of KCH₃COO. The blue circles are the experimental data (47), and the red dashed line is calculated using the relevant Pitzer equation (48). To see this figure in color, go online.

Figure 2

Example simulation box, of the halophilic ferredoxin protein (PDB: 1DOI). Dark blue shape represents the New Cartoon representation of protein, pink spheres represent K⁺, green spheres represent Cl⁻, and transparent small blue dots represent water molecules. To see this figure in color, go online.

Figure 3

(a–c) Molar solution activity derivative (red points) of potassium acetate solutions as a function of the multiplicative scaling factor ($f_{R_{\text{min},{\text{K}}^{+}\text{O}}}$; see Eq. 12) applied to the LJ $R_{\text{min},{\text{K}}^{+}\text{O},\text{LB}}$ parameter governing the interactions between K⁺ and carboxylates. The error bars are the standard error of the mean calculated from three independent production simulations. The red lines are a guide to the eye. The green line shows the experimental reference value; see also Table 2. The area between the two horizontal dashed lines regions show the ±7% deviation from the experimental value. The two vertical dashed lines delimit the range of scaling factors acceptable for the three concentrations. (d) RDF of potassium and the carbon bonded to the oxygens of acetate (the same simulations as in a) for the indicated values of the scaling factor $f_{R_{\text{min},{\text{K}}^{+}\text{O}}}$. To see this figure in color, go online.

Figure 4

(a) Crystal structure (PDB: 1DOI) of the halophilic 2Fe-2S ferredoxin from Haloarcula marismortui (24); the K⁺ ions are shown in pink. The five acidic amino acid sites that have nearby K⁺, used to parameterize $R_{\text{min},{\text{K}}^{+}\text{O}}$, are indicated. (b) Example RDF of K⁺ and the carboxylate oxygens of site 2 at T = 298 K and c_KCl = 1 mol/dm³. The distance to the first maximum is identified as r_sim.. The legend shows the values of the scaling factor $f_{R_{\text{min},{\text{K}}^{+}\text{O}}}$. (c) Unsigned relative deviation between r_sim and r_cryst for the five indicated sites of the halophilic ferredoxin. The protein site is identified by the residue number, residue name, and oxygen name. The legend shows the values of the scaling factor $f_{R_{\text{min},{\text{K}}^{+}\text{O}}}$. Numerical data are shown in Table S1. To see this figure in color, go online.

Figure 5

Surface density of water-protein hydrogen bonds for the indicated halophilic-mesophilic proteins, identified by their pdb ID, for different KCl concentrations. To see this figure in color, go online.

Figure 6

(a and b) Proximal number density of water molecules as a function of the distance to the surface of the indicated proteins, simulated at b_KCl = 2 mol/kg (color) and b_KCl = 0.15 mol/kg (dashed black lines). (c) Height of the first peak of the number density curves for b_KCl = 2 mol/kg, shown in the other panels, as a function of the surface density of acidic amino acids. Each color corresponds to a protein, identified by its pdb ID in the legend of the bottom panel. To see this figure in color, go online.

Figure 7

(a and b) Proximal number density of K⁺ as a function of the distance to the surface of the indicated proteins in solutions with the indicated molality of KCl. (c) Height of the first peak of the number density curves for b_KCl = 2 mol/kg shown in the other panels as a function of the protein charge density. Each color corresponds to a protein, identified by its pdb ID in the legend of the bottom panel. To see this figure in color, go online.

Figure 8

Diffusion coefficients of water around the indicated proteins, simulated in b_KCl = 2 mol/kg. The first shell consists of water molecules that, at t = 0, belong to the first hydration shell of the proteins; bulk consists of all water molecules in the same simulation. To see this figure in color, go online.

Figure 9

Diffusion coefficients of potassium ions around the indicated proteins, simulated in b_KCl = 2 mol/kg. The first shell consists of potassium ions that, at t = 0, belong to the first solvation shell of the proteins; bulk consists of all potassium ions in the same simulation. To see this figure in color, go online.