An osmolyte mitigates the destabilizing effect of protein crowding

Abstract

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.

Keywords: amide proton exchange; macromolecular crowding; nonspecific interaction; osmolytes; protein stability.

Figure 1

Backbone of CI2 colored by stability changes in kcal/mol. (a) in buffered 100.0 g/L protein lysate minus in buffer alone. (b) in buffered 100.0 g/L protein lysate with 0.4 M glycine betaine minusin buffered 0.4 M glycine betaine.

Figure 2

Stability changes brought about by buffered (50 mM sodium phosphate) 100.0 g/L protein lysate containing 0.4 M GB (red; 20°C, pH 7.0) and buffered 100.0 g/L polyvinylpyrrolidone (black, 37°C, pH 5.4, 50 mM sodium acetate) compared to their respective buffers. Positive values denote increased stability. Experiments with only GB were performed once. Bars represent standard errors of the mean for solutions containing 100.0 g/L protein. The PVP data have been published.

Figure 3

Weighted chemical shift changes (Δδ_av) of CI2 compared to buffer [red, 100.0 g/L protein lysate; blue, 100.0 g/L protein lysate plus 0.4 M GB]. Δδ_av is the shift in lysate minus that in buffer. Values greater than 0.02 ppm are significant as shown from replicate experiments.