Thermodynamics of interactions of urea and guanidinium salts with protein surface: Relationship between solute effects on protein processes and changes in water-accessible surface area

Protein noncovalent processes (including solubility or crystallization, conformational changes, ligand binding, and association) fundamentally involve large changes in water-accessible surface area (ASA) and, hence, large changes in the interactions of proteins with water and solutes. It is therefore not surprising that most solutes (at molal concentrations) perturb these processes strongly, even though they are not ligands or direct stoichiometric participants. The direction and extent of perturbation vary with the nature and concentration of the solute. Given the explosion in structural information, it is of great scientific and practical interest to obtain a structural interpretation of solute effects on noncovalent processes involving proteins, nucleic acids, or other biopolymers, and to be able to predict these effects from structural input. To this end, we recently derived a general, quantitative relationship between the effects of charged or uncharged solutes on the standard free energy change ΔGoobs or equilibrium constant Kobs of a biopolymer process and the change in biopolymer ASA (ΔASA) that occurs in that process (Courtenay et al. 2000b; see equation 5 below). This derived relationship between thermodynamic observables and ΔASA complements the empirical relationship between heat capacity changes (ΔCoP,obs and ΔASA of protein processes (Spolar et al. 1989, 1992; Makhadatze and Privalov 1990; Livingstone et al. 1991; Murphy et al. 1992, 1993; Spolar and Record 1994; Myers et al. 1995). Spolar and Record (1994) used ΔCoP,obs for a set of protein–protein and protein–ligand interactions, studied both structurally and thermodynamically, to determine whether these processes were rigid-body interactions or involved significant coupled conformational changes (e.g., folding). Analogous comparisons can be made between estimates of ΔASA determined from the effect of urea on the association process and those predicted from the structural data (assuming a rigid body interaction). The relationship between the effect of a solute on ΔGoobs or Kobs and ΔASA requires knowledge of local-bulk partition coefficients KP,3 for the solutes and the biopolymer surface involved in the process. KP,3 is defined as the ratio of molal concentrations of the perturbing solute in the local domain near the biopolymer surface and in the bulk solution. In the local-bulk domain model, the bulk domain has the same small solute concentration as the two-component (−biopolymer) solute concentration in dialysis equilibrium with the three-component (+biopolymer) solution (Courtenay et al. 2000a,2000b): (1) KP,3 is independent of solute concentration and is greater than unity for accumulated solutes and less than unity for excluded solutes. The purpose of the present work is to determine model-independent thermodynamic preferential interaction coefficients (Γμ3, defined below) that quantify the extent of interaction of protein-destabilizing solutes with native protein surface. Values of KP,3 describing the accumulation of these solutes near native BSA surface are then calculated from Γμ3 using the local-bulk domain model (see below) (Record and Anderson 1995 ; Courtenay et al. 2000a,2000b). The most extensively studied solute effects on any protein process are the effects of urea and guanidinium chloride (GuHCl) on the unfolding of globular proteins and α-helical peptides. The observable in these experiments is the m-value, defined as the negative of the slope of the relatively linear plot of ΔGoobs versus C3, the molar denaturant concentration: (2) Myers et al. (1995) observed that m-values for protein unfolding are proportional to ΔASA, the change in protein ASA on unfolding (empirically corrected for effects of disulfides). Scholtz et al. (1995) and Smith and Scholtz (1996) investigated effects of urea and GuHCl on the stability of a series of chain lengths of alanine-based α-helical peptides, and found that peptide m-values were proportional to chain length. From calculations of the amount and composition of the ASA per residue in the α-helix and completely unfolded states (cf. Fig. 1 ▶), we found that the proportionality constant relating m-values to the ΔASA for unfolding these α-helices is approximately four times larger than that characteristic of the globular proteins studied by Myers et al. (1995). Fig. 1. Decomposition of water-accessible surface areas of native and unfolded proteins and alanine-based peptides and of the surface area exposed on unfolding proteins and alanine-based peptides into contributions from polar peptide backbone, charged, other ... Courtenay et al. (2000b) applied a rigorous thermodynamic analysis to the m-value/ΔASA ratios, taking all significant sources of nonideality in these multicomponent solutions into account, and thereby showed that ΔΓμ3, the difference in denaturant-protein preferential interaction coefficients characteristic of unfolded and folded protein states, is proportional to ΔASA at any specified solute concentration. We found that the dependence of ΔΓμ3/ΔASA on the concentration of urea or GuHCl was quantitatively described by the local-bulk domain model (cf. equation 5 below) and obtained concentration-independent partition coefficients (KunfP,3, listed in Table 1​1)) characterizing the interactions of urea and GuHCl with the protein and peptide surfaces exposed on unfolding. Table 1. Partition coefficients for urea and guanidinium salts Figure 1 ▶ illustrates the key differences in the composition of water-accessible surface calculated for native BSA and the average globular protein (Miller et al. 1987) and for the protein or peptide surface exposed in unfolding. In particular, the surface exposed on unfolding differs significantly from native protein surface because of the virtual absence of any contribution from charged residues, which are water accessible in native and denatured states. Moreover, 48% of the surface exposed on unfolding the alanine-based α-helices is polar peptide backbone, whereas this subclass of polar surface constitutes only 13% of the ΔASA of unfolding of globular proteins (and of the ASA of native globular proteins). Courtenay et al. (2000b) observed that the four-fold greater percentage contribution of polar peptide surface to the ΔASA of unfolding of α-helical peptides as compared to globular proteins matched the four-fold higher m-value: ΔASA ratio observed for the α-helical peptides. This correspondence provides independent evidence that the primary region of interaction of urea or GuH+ with the protein is the polar peptide backbone, the conclusion obtained in many previous studies of transfers of model peptides from water to solutions of urea (Gill et al. 1961; Nozaki and Tanford 1963; Robinson and Jencks 1965a; Nandi and Robinson 1984; Sijpkes et al. 1993; Wang and Bolen 1997) and GuHCl (Castellino and Barker 1969; Robinson and Jencks 1965a; Nozaki and Tanford 1970; Nandi and Robinson 1984). In the present study we report model-independent preferential interaction coefficients Γμ3 calculated from osmometric data on interactions of three protein destabilizing solutes (urea, GuHCl, and guanidinium thiocyanate, i.e., GuHSCN) and the reference solute KCl with native BSA. BSA is a member of the homologous series of native proteins that differ in total ASA but have similar surface compositions (see Fig. 1 ▶). In particular, native BSA has the same percentages of nonpolar and polar surface as the average globular protein. Within the class of polar surface, BSA has the same percentage of polar backbone surface (13%), but a larger percentage of charged surface (29% vs. 19%). We interpret values of Γμ3 as a function of small solute concentration using the local-domain model to obtain partition coefficients characterizing the accumulation of these solutes near the surface of native BSA relative to their concentrations in the bulk solution. We compare these partition coefficients with those determined by Courtenay et al. (2000b) for the interactions of these solutes with the protein surface exposed on unfolding globular proteins and α-helices. The comparisons presented here between interactions with native and with unfolded protein surfaces are relevant for the mechanism of action of these denaturants, and provide further evidence that urea and GuH+ unfold proteins primarily by their favorable interaction (local accumulation) with the polar peptide surface that is exposed on unfolding. Our quantitative characterization of interactions of GuH+, Cl− and SCN− with protein surface is a useful starting point for predicting Hofmeister effects of these and other ions on protein processes from structural information (ΔASA).