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

Abstract

To interpret effects of urea and guanidinium (GuH(+)) salts on processes that involve large changes in protein water-accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Gamma(mu3)) as functions of nondenaturing concentrations of these solutes (0-1 molal). From analysis of Gamma(mu3) using the local-bulk domain model, we obtain concentration-independent partition coefficients K(nat)(P) that characterize the accumulation of these solutes near native protein (BSA) surface: K(nat)(P,urea)= 1.10 +/- 0.04, K(nat)(P,SCN(-)) = 2.4 +/- 0.2, K(nat)(P,GuH(+)) = 1.60 +/- 0.08, relative to K(nat)(P,K(+)) identical with 1 and K(nat)(P,Cl(-)) = 1.0 +/- 0.08. The relative magnitudes of K(nat)(P) are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (K(nat)(P)) with those determined by us previously for unfolded protein and alanine-based peptide surface K(unf)(P), we dissect K(P) into contributions from polar peptide backbone and other types of protein surface. For globular protein-urea interactions, we find K(nat)(P,urea) = K(unf)(P,urea). We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of K(P) and the change in protein ASA.

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 polar, and nonpolar surface (see Materials and Methods).

Fig. 2.

Solution osmolality as a function of solute molality for solutions of protein destabilizing solutes with (filled symbols) and without (open symbols) 3.16 mM BSA. (A): urea, (B): GuHCl, and (C): GuHSCN solutions.

Fig. 3.

Change in solution osmolality (ΔOsm_ex) in excess of that expected assuming additivity on addition of BSA (3×10⁻³ molal final concentration) as a function of two-component (small solute-water) solution osmolality. Uncertainties in calculated values of ΔOsm_ex are approximately ±15%.

Fig. 4.

Preferential interaction coefficients (Γ_μ3) for native BSA-destabilizing solute interactions as a function of bulk solute molality (m₃^bulk) calculated as described by Courtenay et al. (2000a). Panels A–C show all data for urea (at 2.70, 3.16, and 3.61 mM BSA), GuHCl (at 2.90 and 3.16 mM BSA), and GuHSCN (at 2.56, 3.16, and 3.75 mM BSA). Error bars represent the propagated experimental uncertainty. No dependence of Γ_μ3 on BSA concentration was observed.

Fig. 5.

Quantitative solute series tabulating local-bulk partition coefficients K_P for the interactions of cations, anions, and uncharged or zwitterionic solutes with native protein surface and with the surface exposed on unfolding proteins or alanine-based peptides. Ion partition coefficients are calculated based on the assignment K_P,K⁺ ≡ 1 and the assumption that K_P,Cl⁻ = 1 has the same value for the surface exposed on unfolding as for native BSA surface (K^nat_P,KCl = 1.00 ± 0.08).

Fig. A1.

KCl-BSA interactions. Panel A contains representative VPO data showing the dependence of solution osmolality on KCl molality in the presence (filled circles) and absence (open circles) of 3.88 mM BSA. Panel B plots Γ_μ3 as a function of bulk KCl molality (at 3.07, 3.51, and 3.88 mM BSA), calculated as described previously (Courtenay et al. 2000a). For this system, the two approximations (see Courtenay et al. 2000a) give equivalent determinations of Γ_μ3 for the entire concentration range presented. No BSA concentration dependence of Γ_μ3 as a function of bulk KCl concentration is observed.

Fig. A2.

Plots of Γ_μ3 for interactions of BSA with protein-destabilizing GuH⁺ salts as a function of bulk solute molality (m₃^bulk). Panels A and B show data for GuHCl and GuHSCN in which Γ_μ3 has been calculated using either Approximation I, μ₂₂ = μ^o₂₂ (circles), or Approximation II, μ₃₃ = μ^o₃₃ (squares) (Courtenay et al. 2000a). The triangle represents the preferential interaction coefficient for GuHCl-BSA interactions determined by equilibrium dialysis at m₃^bulk = 1 molal (Arakawa and Timasheff 1984a).