Sugar osmolytes-induced stabilization of RNase A in macromolecular crowded cellular environment

Organisms synthesize sugar osmolytes during environmental stresses to protect proteins against denaturation. These studies were carried out in dilute buffer whereas intracellular milieu within cells has cytoplasmic concentration of macromolecules in the range of 80-400 mg ml^-1. Is the stabilizing effect of sugar osmolytes on the protein in dilute buffer different from that when protein is in cellular environment? To answer this question, we have measured and analysed the effect of sugar osmolytes on the structural and thermodynamic stability of ribonuclease A in the presence of dextran 70 at multiple concentrations of six sugars at different pH values. It was found that (i) each sugar osmolyte in the crowded environment provides stability to the protein in terms of T_m (midpoint of denaturation) and ∆G_D° (Gibbs energy change) and this stabilizing effect is under entropic control, (ii) the extent of osmolyte-induced stabilization of RNase A is pH dependent, and (iii) effect of sugars on the stability of protein in presence of the crowding agent remains unchanged. This study concludes that crowding does not affect the efficacy of osmolytes and vice versa; and emphasizes on understanding of internal architecture of the cellular environment with respect to molecular and macromolecular crowding.