Effect of Salts on the Stability and Folding of Staphylococcal Nuclease

Abstract

The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.

Atypical Effect of Salts on the Thermodynamic Stability of Human Prion Protein

“…The observed decrease in the thermodynamic stability of huPrP90 -231 in the presence of sodium fluoride, sodium sulfate, sodium acetate, and sodium chloride is highly unusual because these salts are generally known to have a stabilizing effect on proteins (33)(34)(35)(36). To gain insight into the molecular basis of this effect, measurements similar to those described above were performed at neutral pH.…”

Protein Stabilization by Osmolytes from Hyperthermophiles

“…independently of pH and the protein surface charge, and usually requires high concentrations to be effective. Fewer studies have been performed with charged solutes, but the ability of certain ions to bind and stabilize proteins has been reported, and it is generally believed that direct protein-solute interactions play a relevant role in the mechanism of protein stabilization by salts (7)(8)(9)(10). Excellent reports on the principles that control protein stabilization by salts and electrically neutral molecules are available (4 -6, 11-13).…”

Salt Modulates the Stability and Lipid Binding Affinity of the Adipocyte Lipid-binding Proteins

“…1, shows that large patches or clusters of like charge occur on the surface of the protein. As has been proposed by Fink and associates (33,37), added salt would screen the repulsive interactions within each extended patch and thus stabilize the protein.…”