Perturbed pKA-values in the denatured states of proteins.

We show in this study that the ionisation equilibria of denatured proteins in pure water are inconsistent with the "fully-unfolded" conformation being an extended coil where the residues are isolated from one another by the intervening solvent. The effects of acid and salt on the stability of the barley chymotrypsin inhibitor 2 (CI2) were investigated and the pKA-values of all carboxylate residues in the native protein were determined by NMR. A comparison of the experimentally determined pH-dependence of the protein stability and that calculated using observed pKA-values in the native state, reveals that the pKA-values in the denatured state are, on average, 0.3 pH units lower than those of model compounds. An increase in ionic strength eliminates these pKA shifts in the denatured state. This shows that there are electrostatic interactions in the denatured state of CI2. Since previous studies on barnase and the Ovomucoid Third Domain also report anomalous titration behaviours of the denatured states, it appears that perturbed pKA-values in the denatured state is a general phenomenon, indicating that the unfolded conformation in pure water is a fairly compact species. In addition, we used a mutational approach to determine the pKA-values of a carboxylate group in both the native and denatured states. The pKA-value in the native state obtained by this method is in precise agreement with that obtained by NMR.