It has long been known that large soft anions like bromide, iodide and thiocyanate are protein denaturing agents, but their mechanism of action is still unclear. In this work we have investigated the protein denaturing properties of these anions using Ribonuclease A (RNase A) as a model protein system. Salt-induced perturbations to the protein folding free energy were determined using differential scanning calorimetry and the results demonstrate that the addition of sodium iodide and sodium thiocyanate significantly decreases the melting temperature of the protein. In order to account for this reduction in protein stability, we show that the introduction of salts that contain soft anions to the aqueous solvent perturbs the protein unfolding free energy through three mechanisms: (a) screening Coulomb interactions that exist between charged protein residues, (b) Hofmeister effects, and (c) specific anion binding to CH and CH2 moieties in the protein polypeptide backbone. Using the micellization of 1,2-hexanediol as a ruler for hydrophobicity, we have devised a practical methodology that separates the Coulomb and Hofmeister contributions of salts to the protein unfolding free energy. This allowing us to isolate the contribution of soft anion binding interactions to the unfolding process. The analysis shows that binding contributions have the largest magnitude, confirming that it is the binding of soft anions to the polypeptide backbone that is the main promoter of protein unfolding.
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