Effects of macromolecular crowding on the structural stability of human α-lactalbumin

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments, α-Lactalbumin, as a model system for proteinolding studies, has been used extensively because it can form stable molten globule states under a range of conditions. Here we report that the crowding agents Ficoll 70, dextran 70, and polyethylene glycol （PEG） 2000 have different effects on the structural stability of human α-lactalbumin （HLA） represented by the transition to a molten globule state： dextran 70 dramatically enhances the thermal stability of Ca^2＋-depleted HLA （apo-HLA） and Ficoll 70 enhances the thermal stability of apo-HLA to some extent, while PEG 2000 significantly decreases the thermal stability of apo-HLA. Ficoll 70 and dextran 70 have no obvious effects on trypsin degradation of apo-HLA but PEG 2000 accelerates apo-HLA degrad- ation by trypsin and destabilizes the native conformation of apo-HLA. Furthermore, no interaction is observed between apo-HLA and Ficoll 70 or dextran 70, but a weak, non-specific interaction between the apo form of the protein and PEG 2000 is detected, and such a weak, non-specific interaction could overcome the excluded- volume effect of PEG 2000. Our data are consistent with the results of protein stability studies in cells and suggest that stabilizing excluded-volume effects of crowding agents can be ameliorated by non-specific interactions between proteins and crowders.