Effects of sugars and polyols on the stability of azurin in ice

This study represents the first attempt to gain a quantitative estimate of the protective influence of sugars (sucrose and trehalose) and polyols (sorbitol and glycerol) on the thermodynamic stability (Delta G degrees) of a protein in low-temperature part-frozen aqueous solutions. The method, based on guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, distinguishes between the effects of cooling to subfreezing temperatures from those induced specifically by the formation of a solid ice phase. The results point out that in the liquid state the generally stabilizing effect (at molar concentrations) of these polyhydric compounds is markedly attenuated on cooling to subfreezing temperatures such that at -15 degrees C, only sucrose still exerts a significant increase in Delta G degrees. At this temperature, and in the absence of additives, the formation of ice caused a progressive destabilization of the native fold, Delta G degrees decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice (V-L) was reduced to less than 1%. Unexpectedly, denaturation profiles in ice at selected V-L demonstrate that none of the above sugars and polyols counters effectively the decrease in protein stability at small V-L. Only trehalose was able to partly attenuate the ice perturbation, raising Delta G degrees by a modest 0.6-0.8 kcal/mol relative to the salt reference. In all cases the reduction in Delta G degrees caused by the solidification of water correlates with the decrease in m-value. The implication is that Delta ASA of unfolding is smaller in ice because protein-ice interactions either increase the solvent-accessible surface area (ASA) of the native fold (partial unfolding) or reduce the ASA of the denatured state (compaction), or both. Information on the protein tertiary structure in ice, in the absence and in the presence of sucrose or glycerol, suggests that these osmolytes play an important role in maintaining a compact native state that in their absence is expanded and partly unfolded. Thus, it appears that the prevailing mechanism by which these osmolytes act as cryoprotectants is through preservation of the native conformation in the liquidus rather than by increasing the thermodynamic stability of the native fold.