Polyols and sugars stabilize diverse and specific structural regions of beta-lactoglobulin, a milk allergen ,against temperature-induced denaturation

Thermal treatments of foods should be tuned in order to optimize the balance between nutritional, nutraceutical and antinutritional factors. In this frame, temperature-induced unfolding has been used to decrease immunoreactivty of allergenic food proteins, either by directly affecting conformational epitopes or by promoting protease action on "buried" sequential epitopes. However, in this frame, most theoretical and experimental studies have considered only pure and isolated molecules, without taking into account that many common food ingredients may have drastic effects on the thermal stability of protein structures. Here we report a study on the temperature sensitivity of selected structural features of bovine milk betalactoglobilin (BLG), an allergenic milk protein, in the presence of two common food ingredients (sucrose and sorbitol) or two reference molecules (glycerol and trehalose). None of them affected the structural features of the protein at room temperature, where the only observed effect was an increased affinity towards hydrophobic molecules in the presence of all co-solutes but glycerol. Temperature-ramp approaches were used to study the effects of co-solutes on the stability of the beta-barrel region of BLG by circular dichroism measurements, and to measure the temperature dependence of the exposure of the Cys121 thiol to suitable reagents as a consequence of movement of the C-terminal helix of BLG away from the beta-barrel region. These events are known to have reportedly different temperature dependence. The efficiency of co-solutes at stabilizing each of the two regions was different, suggesting that each of them acts in a specific way on the solvent/protein system. Differential scanning calorimetry study indicated that most of the observed stabilization - in particular in the case of the largest and most solvated co-solutes - is due to entropic effects. Thus, although results are fitting within the "preferential exclusion" theory of protein stabilization, this study points out that different structural regions of the same protein have a different and specific response to individual co-solutes.