Dissecting the stabilizing effects of polyols on protein structure

Polyols have long been known to impact stability of proteins folding. Their stabilizing effect is widely exploited in pharmacological preparation and has repercussion also in food science. Despite this, their exact mechanism of action is not well understood. Beta-lactoglobulin (BLG) is a protein with a well know denaturation behaviour, also proposed as a drug carrier. In this study BLG was used to assess the effects of high concentrations of sucrose or sorbitol (20 and 40% w/v) on the unfolding of various structural regions in BLG. Both polyols do not affect the functional properties of BLG, as the ligand binding ability of the protein is preserved even at the highest polyol concentration. Both sucrose and sorbitol stabilize the overall fold of BLG against physical denaturants, as assessed by near-UV circular dichroism, and that of specific regions near the surface helix, as assessed by reactivity of the hidden Cys121. The stabilizing effect is proportional to the concentration of the co-solute. Only sucrose stabilization of the helix structure shows a saturation effect, suggesting a different mechanism of action for sucrose. Indeed, 40% sucrose also affects the dissociation equilibrium of the BLG native dimer, as calculated from the diffusion coefficient determined by NMR spectroscopy.