Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol

Abstract

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Effect of glycerol and dimethyl sulfoxide on the phase behavior of lysozyme: Theory and experiments

“…Its increase with increasing temperature and glycerol content is consistent with previous findings. 12,20,22 As mentioned above, glycerol affects b 2 (T) less than DMSO, i.e., the addition of 10 vol.% glycerol has about the same effect as 5 vol.% DMSO (Figs. 6(b) and 6(c)), although the optical properties of the two solvents suggest a similar effect on A ν > 0 and thus A (Fig.…”

“…For the additive concentrations studied here, lysozyme does not unfold or undergo conformational changes. [11][12][13] This allows us to test the validity and limits of the simple model, to investigate the effects of these additives on the interactions and the phase behavior, and to understand at least qualitatively the trends observed in our experiments.…”

“…20,21 In the presence of glycerol, Esposito et al 22 furthermore observed an increase of the hydrodynamic radius with increasing temperature. Using computer simulations, Vagenende et al 12 found that glycerol preferentially interacts with the hydrophobic patches on the lysozyme surface. Glycerol molecules shield these patches with their hydrocarbon backbone, allowing the lysozyme hydration shell to grow.…”

“…This is in accordance with recent computer simulation results. 12 Having determined the values of all parameters, we calculated the interaction potential for different temperatures and compositions, added salt (NaCl) concentrations as well as different glycerol and DMSO contents. We found that glycerol and DMSO increase the repulsive interaction, as quantified by the second virial coefficient b 2 .…”

Fibers Obtained from Alginate, Chitosan and Hybrid Used in the Development of Scaffolds

“…Compared to the scaffolds without glycerol, the scaffolds containing glycerol presented less weight loss in all scaffolds, suggesting that the presence of glycerol worked as a stabilizer of the scaffold structures 36,37 .…”