Thermodynamic analysis of interactions between denaturants and protein surface exposed on unfolding: Interpretation of urea and guanidinium chloride m-values and their correlation with changes in accessible surface area (ASA) using preferential interaction coefficients and the local-bulk domain model

A denaturant m-value is the magnitude of the slope of a typically linear pl ot of the unfolding free energy change dG(obs)degrees vs. molar concentrati on (C-3) of denaturant, For a given protein, the guanidinium chloride (GuHC l) m-value is approximately twice as large as the urea m-value. Myers et al . (Protein Sci 1995;4:2138-2148) found that experimental m-values for prote in unfolding in both urea and GuHCl are proportional to Delta ASA(corr)(max ), the calculated maximum amount of protein surface exposed to water in unf olding, corrected empirically for the effects of disulfide crosslinks: (ure a m-value/Delta ASA(corr)(max)) = 0.14+/-0.01 cal M-1 Angstrom(-2) and (GuH Cl m-value/Delta ASA(corr)(max)) = 0.28+/-0.03 cal M-1 Angstrom(-2). The observed linearity of plots of Delta G(obs)degrees vs, C-3 indicates th at the difference in preferential interaction coefficients Delta Gamma(3) c haracterizing the interactions of these solutes with denatured and native p rotein surface is approximately proportional to denaturant concentration. T he proportionality of m-values to Delta ASA(corr)(max) indicates that the c orresponding Delta Gamma(3) are proportional to Delta ASA(corr)(max) at any specified solute concentration. sere we use the local-bulk domain model of solute partitioning in the protein solution (Courtenay et al., Biochemistr y 2000;39:4455-4471) to obtain a novel quantitative interpretation of denat urant m-values, We deduce that the proportionality of m-value to Delta ASA( corr)(max) results from the proportionality of B-1(0) (the amount of water in the local domain surrounding the protein surface exposed upon unfolding) to Delta ASA(corr)(max). We show that both the approximate proportionality of Delta Gamma(3), to denaturant concentration and the residual dependence of Delta Gamma(3)/m(3) (where m(3) is molal concentration) on denaturant c oncentration are quantitatively predicted by the local-bulk domain model if the molal-scale solute partition coefficient K-P and water-solute exchange stoichiometry S-1,S-3 are independent of solute concentration. We obtain K -P,K-urea = 1.12+/-0.01 and K-P,K-GuHcl = 1.16+/-0.02 (or R-P,R-GuH+ congru ent to 1.48), values which will be useful to characterize the effect of acc umulation of those solutes on all processes in which the water-accessible a rea of unfolded protein surface changes. We demonstrate that the local-bulk domain analysis of an m-value plot justifies the use of linear extrapolati on to estimate (less than or similar to 5% error) the stability of the nati ve protein in the absence of denaturant (Delta G(o)(o)), with respect to a particular unfolded state. Our surface area calculations indicate that published m-values/Delta ASA ra tios for unfolding of alanine-based alpha-helical oligopeptides by urea and GuHCl exceed the corresponding m-value/Delta ASA ratios for protein unfold ing by approximately fourfold, We propose that this difference originates f rom the approximately fourfold difference (48% vs. 13%) in the contribution of polar backbone residues to Delta ASA of unfolding, a novel finding whic h supports the long-standing but not universally accepted hypothesis that u rea and guanidinium cations interact primarily with backbone amide groups. We propose that proteins which exhibit significant deviations from the aver age m-value/Delta ASA ratio will be found to exhibit significant deviations from the expected amount and/or average composition of the surface exposed on unfolding, (C) 2000 Wiley-Liss, Inc.