Apparent p Ka shifts of titratable residues at high denaturant concentration and the impact on protein stability

Abstract

Urea and guanidine–hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain p K a values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. p K a values of all four residues are up-shifted by 0.3–0.5 pH units in presence of 6 M urea by comparison with p K a values of the residues dissolved in water. In the presence of 6 M GdnHCl, p K a values are down-shifted by 0.2–0.3 pH units in the case of acidic and up-shifted by 0.3–0.5 pH units in the case of basic residues. Shifted p K a values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.