PERTURBED PK(A)-VALUES IN THE DENATURED STATES OF PROTEINS

We show in this study that the ionisation equilibria of denatured prot eins in pure water are inconsistent with the ''fully-unfolded'' confor mation being an extended coil where the residues are isolated from one another by the intervening solvent. The effects of acid and salt on t he stability of the barley chymotrypsin inhibitor 2 (CI2) were investi gated and the pK(A)-values of all carboxylate residues in the native p rotein were determined by NMR. A comparison of the experimentally dete rmined pH-dependence of the protein stability and that calculated usin g observed pK(A)-values in the native state, reveals that the pK(A)-va lues in the denatured state are, on average, 0.3 pH units lower than t hose of model compounds. An increase in ionic strength eliminates thes e pK(A) shifts in the dentured state. This shows that there are electr ostatic interactions in the denatured state of CI2. Since previous stu dies on barnase and the Ovomucoid Third Domain also report anomalous t itration behaviours of the denatured states, it appears that perturbed pK(A)-values in the denatured state is a general phenomenon, indicati ng that the unfolded conformation in pure water is a fairly compact sp ecies. Ln addition, we used a mutational approach to determine the pK( A)-values of a carboxylate group in both the native and denatured stat es. The pK(A)-value in the native state obtained by this method is in precise agreement with that obtained by NMR. (C) 1995 academic Press L imited