Determinants of increased stability of the beta-lactoglobulin folding in the presence of polyols

Stability of protein folding in systems that include high concentrations of non-ionic solutes is of great practical relevance to the food and biotechnology fields. The unfolding of bovine betalactoglobulin (BLG) has been much studied, also because this food protein is a major food allergen and a vehicle for a number of micronutrients. Unfolding of various structural regions in BLG was monitored by means of various spectroscopic techniques in the presence/absence of sucrose or sorbitol (20 to 40% by weight). At room temperature, the added agents altered the 1H NMR signals from Glu89 (involved in the Tanford transition and in controlling access to the binding cavity in BLG), although they did not affect the binding of hydrophobic molecules. Temperature dependence studies indicated that sugars and polyols stabilized against: 1) breakdown of the hydrophobic contacts that ensure interaction of the C-terminus helix with the beta-barrel main protein body (this is essential to prevent polymerization of the unfolded protein through disulfide exchange); 2) overall loss of tertiary structure, as monitored by near-UV CD; 3) formation of covalent and non-covalent aggregates among partially denatured BLG isoforms, as aggregation was remarkably slowed down by the added agents. Thus, individual steps of BLG unfolding (and of ensuing aggregation) appear to be specifically affected by the additions of either sugars or polyols.