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.

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.

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 plot of the unfolding free energy change DeltaG degrees (obs) vs. molar concentration (C(3)) 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 DeltaASA(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/DeltaASA(corr)(max)) = 0.14+/-0.01 cal M(-1) A(-2) and (GuHCl m-value/DeltaASA(corr)(max)) = 0.28+/-0.03 cal M(-1) A(-2). The observed linearity of plots of DeltaG degrees (obs) vs. C(3) indicates that the difference in preferential interaction coefficients DeltaGamma(3) characterizing the interactions of these solutes with denatured and native protein surface is approximately proportional to denaturant concentration. The proportionality of m-values to DeltaASA(corr)(max) indicates that the corresponding DeltaGamma(3) are proportional to DeltaASA(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 DeltaASA(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 DeltaASA(corr)(max). We show that both the approximate proportionality of DeltaGamma(3) to denaturant concentration and the residual dependence of DeltaGamma(3)/m(3) (where m(3) 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 S(1,3) 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+) congruent with 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 ( less, similar 5% error) the stability of the native protein in the absence of denaturant (DeltaG(o)(o)), with respect to a particular unfolded state. Our surface area calculations indicate that published m-values/DeltaASA ratios for unfolding of alanine-based alpha-helical oligopeptides by urea and GuHCl exceed the corresponding m-value/DeltaASA 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 DeltaASA of unfolding, a novel finding which supports the long-standing but not universally accepted hypothesis that urea and guanidinium cation interact primarily with backbone amide groups. We propose that proteins which exhibit significant deviations from the average m-value/DeltaASA ratio will be found to exhibit significant deviations from the expected amount and/or average composition of the surface exposed on unfolding.

Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro.

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine >> proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.

Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments.

Escherichia coli adapts to changes in growth osmolarity of at least 100-fold by making large changes in the amounts of intracellular water and solutes, including cytoplasmic K+. A wide range of in vitro salt, solute and biopolymer concentrations should therefore be considered 'physiological'. Paradoxically, these large, osmotically induced changes in cytoplasmic K+ concentration do not greatly affect the equilibria and kinetics of cytoplasmic protein-nucleic acid interactions. Biophysical effects resulting from changes in the amount of cytoplasmic water (such as macromolecular crowding) and in the concentrations of other cytoplasmic solutes appear to compensate for the effects of changes in cytoplasmic K+ concentration and thereby maintain protein-nucleic acid equilibria and kinetics in the range required for in vivo function.

Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water.

Escherichia coli is capable of growing in environments ranging from very dilute aqueous solutions of essential nutrients to media containing molar concentrations of salts or nonelectrolyte solutes. Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiological challenges for cells. To meet these challenges, E. coli adjusts a wide range of cytoplasmic solution variables, including the cytoplasmic amounts both of water and of charged and uncharged solutes.