Polyols stabilize specific regions in the structure of betalactoglobulin

Polyols have long been known to improve the stability of protein folding, but the exact mechanism of their stabilizing action remains debated. In this work, beta-lactoglobulin (BLG) was used to study the mechanism of stabilization for four common polyols: sucrose, sorbitol, glycerol, and trehalose. BLG is an excellent model protein given its well know (and complex) denaturation behavior: unfolding of BLG occur through a number of steps, each affecting separate regions of the protein. Stabilizing effects of various concentrations of different polyols towards physical and chemical denaturation were assessed by near-UV circular dichroism, and by evaluating the reactivity of Cys121, whose thiol is hidden in native BLG. Surface hydrophobic interactions were characterized by using suitable non-covalent fluorescent probes. Effects of polyols on the dissociation equilibrium of the BLG native dimer were assessed by estimating a diffusion coefficient through NMR spectroscopy. The stabilizing effects are discussed in terms of the preferential exclusion theory, and of the effects of individual polyols on solvent properties, in order to point out structure-specific aspects of the overall effect of individual polyols. Polyols in solution shift the Native/Denatured equilibrium towards the native state. Sucrose, sorbitol, and trehalose share many similarities in the mechanism of stabilization: they have the strongest stabilizing properties, that appear related to their molar concentration. On the contrary, glycerol presents peculiar features and the lowest stabilizing effects. The effect of polyols on the overall denaturation of BLG (as detected by thermal analysis) relates to ability of each polyol to improve surface hydrophobic interactions (i.e., within the solvent-excluded region). This interpretation is also supported by our CD data. However, individual polyols have region-specific stabilizing effects, suggesting that each of them has a somewhat specific mechanism of action. In spite of the huge number of literature reports, properties of polyols in solution are not completely understood yet, and polyols are still used on quite empirical basis. This study indicates that stabilizing effects of individual polyols target polyol-specific structural features of the protein, and are not only a function of the polyol(s) physico-chemical features, paving the road to improving their rational application in protein stabilization.