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

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Abstract

A denaturant m-value is the magnitude of the slope of a typically linear plot of the unfolding free energy change ΔG(obs)/°vs. molar concentration (C3) of denaturant. For a given protein, the guanidinium chloride (GuHCl) 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 protein unfolding in both urea and GuHCl are proportional to ΔASA(corr)/(max), the calculated maximum amount of protein surface exposed to water in unfolding, corrected empirically for the effects of disulfide crosslinks: (urea m-value/ΔASA(corr)/(max)) = 0.14±0.01 cal M-1 Å-2 and (GuHCl m-value/ΔASA(corr)/(max)) = 0.28±0.03 cal M-1 Å-2. The observed linearity of plots of ΔG(obs)/°vs. C3 indicates that the difference in preferential interaction coefficients ΔΓ3 characterizing the interactions of these solutes with denatured and native protein surface is approximately proportional to denaturant concentration. The proportionality of m-values to ΔASA(corr)/(max) indicates that the corresponding ΔΓ3 are proportional to ΔASA(corr)/(max) at any specified solute concentration. Here we use the local-bulk domain model of solute partitioning in the protein solution (Courtenay et al., Biochemistry 2000;39:4455-4471) to obtain a novel quantitative interpretation of denaturant m-values. We deduce that the proportionality of m-value to ΔASA(corr)/(max) results from the proportionality of B1/0 (the amount of water in the local domain surrounding the protein surface exposed upon unfolding) to ΔASA(corr)/(max). We show that both the approximate proportionality of ΔΓ3 to denaturant concentration and the residual dependence of ΔΓ3/m3 (where m3 is molal concentration) on denaturant concentration are quantitatively predicted by the local-bulk domain model if the molal-scale solute partition coefficient K(P) and water-solute exchange stoichiometry S1,3 are independent of solute concentration. We obtain K(P,urea) = 1.12±0.01 and K(P,GuHCl) = 1.16±0.02 (or K(P,GuH+) ≃ 1.48), values which will be useful to characterize the effect of accumulation of those solutes on all processes in which the water-accessible area of unfolded protein surface changes. We demonstrate that the local-bulk domain analysis of an m-value plot justifies the use of linear extrapolation to estimate (≤ 5% error) the stability of the native protein in the absence of denaturant (ΔG(o)/(o)), with respect to a particular unfolded state. Our surface area calculations indicate that published m-values/ΔASA ratios for unfolding of alanine-based α-helical oligopeptides by urea and GuHCl exceed the corresponding m-value/ΔASA ratios for protein unfolding by approximately fourfold. We propose that this difference originates from the approximately fourfold difference (48% vs. 13%) in the contribution of polar backbone residues to ΔASA of unfolding, a novel finding which supports the long-standing but not universally accepted hypothesis that urea and guanidinium cations interact primarily with backbone amide groups. We propose that proteins which exhibit significant deviations from the average m-value/ΔASA 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.

Original languageEnglish
Pages (from-to)72-85
Number of pages14
JournalProteins: Structure, Function and Genetics
Volume41
Issue numberSUPPL. 4
DOIs
StatePublished - Oct 26 2000

Keywords

  • Activity coefficient
  • Linear extrapolation model
  • Partition coefficient
  • Peptide backbone
  • Protein stability

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