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

Abstract

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. a-Lactalbumin, as a model system for protein-folding 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 a-lactalbumin (HLA) represented by the transition to a molten globule state: dextran 70 dramatically enhances the thermal stability of Ca 21 -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 degradation 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 excludedvolume 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.

Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins

“…To investigate the possibility that these spectral changes were due to the crowding agents binding to TC-1, we performed isothermal calorimetry (ITC) experiments, titrating 0.1 mM TC-1 into 160 g/L crowder solutions (Figure S3). These measurements were not indicative of specific interactions between TC-1 and Ficoll or Dextran 70 [72].…”

Macromolecular crowding: Macromolecules friend or foe

“…Furthermore, the destabilizing influence of the reconstituted E. coli cytosol on chymotrypsin inhibitor 2 stability was observed to be stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions [44]. In another development [45], PEG 2000 was found to decrease the thermal stability of apo human α-lactalbumin thereby accelerating its degradation by trypsin. Isothermal titration calorimetry (ITC) measurements revealed a weak, nonspecific interaction between the apo form of the protein and PEG 2000 suggesting that the lack of crowding-induced stabilizing influence on protein stability is due to the amelioration of the stabilizing excluded volume effects of crowding agents by nonspecific interactions between protein and crowder.…”

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

“…In addition to the effect of excluded volume, the presence of a crowding agent in a solution may affect the properties of a tested protein in some other ways. In the presence of some direct interactions of a target protein with a crowding agent, the excluded volume effects can be either strengthened or weakened [42,43]. …”