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 Tm (midpoint of denaturation) and ∆GD° (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.

Proteins in the insulin-secreting cell line MIN6 bind the imidazoline compound BL11282.

The imidazoline BL11282 stimulates insulin release and alters islet proteomes. Subcellular fractions of MIN6 cells showed that the membrane fraction exhibited binding to BL11282 on a Biacore chip and to BL11282-labelled magnetic beads. Bound material extracted from the beads showed a approximately 50 kDa differential band upon SDS-PAGE and a weaker 100 kDa band. The former was sensitive to competitive removal by preincubation of the fraction with BL11282, then highlighting the approximately 100 kDa band. Masspectrometric analysis revealed the approximately 50 kDa band to be EF1A and the approximately 100 kDa band to be glucose regulated P94, both of interest in insulin synthesis and secretion.