Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins

Abstract

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native-state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native-state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far- and near-UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native-state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl-induced unfolding, increased in proportion to sucrose concentration. Native-state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange-resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large-scale than on small-scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native-state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.

Figure 1.

The far- (A–C) and near-UV (D–F) CD spectra of RNase A (A,D), RNase S (B,E), and Cyt c (C,F) in the absence (solid lines) and presence (dashed-dotted lines) of 1 M sucrose at 25°C.

Figure 2.

GdnHCl-induced unfolding curves for RNase A (A), RNase S (B), and Cyt c (C) in 0 (○), 0.5 (▪), and 1 M (▵) sucrose concentrations. Error bars represent the standard deviation for triplicate samples.

Figure 3.

Thermal-induced unfolding curves of RNase A (A), RNase S (B), and Cyt c (C) as a function of sucrose concentration; for RNase A and S, 0 (○), 0.5 (▪), and 1 M (▵) sucrose concentrations; for Cyt c, 0 (•) and 0.75 M (○) sucrose concentrations. The solid lines in A and B represent the fitted curve by the complex, nonlinear least-squares equation to the raw data.

Figure 4.

Representative IR amide absorbance spectra of HX for RNase A (A), RNase S (B), and Cyt c (C), showing the amide I, II, and II′ peaks. The solid and dotted lines represent the spectra of nondeuterated proteins in H₂O buffer and the completely deuterated proteins in 100% D₂O buffer, respectively. Other lines are representative spectra of proteins exposed to 75% D₂O buffer. Arrows indicate the direction of spectral changes with the progress of HX.

Figure 5.

The fraction of unexchanged amide hydrogens of RNase A (A), RNase S (B), and Cyt c (C) in the presence (○) and absence (•) of 1 M sucrose. The solid lines represent the fitting curve with equation 3. Error bars indicate the standard deviation for triplicate samples of RNase A and S and for duplicate samples of Cyt c.

Figure 6.

The HX rate constant as a function of X for RNase A (A), RNase S (B), and Cyt c (C) in the presence (○) and absence (•) of 1 M sucrose.

Figure 7.

The coupling factor, f(X), between effects of sucrose on HX and complete unfolding as a function of the fraction of unexchanged amide hydrogens, X, for RNase A (•) and RNase S (○).