Glycine Betaine Reverses Osmotic Shock Induced Protein Destabilization in Living Cells

Abstract

Cells utilize several mechanisms for adapting to changes in osmotic pressure. Bacteria, including Escherichia coli, can grow in a wide range of osmolarities. Increasing the external osmolarity (i.e., hyperosmotic shock) causes water efflux, reduction in cell volume and accumulation of osmolytes such as glycine betaine. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living E. coli cells before and after osmotic shock in the presence and absence of glycine betaine. The 7-kDa N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) is used as the model protein because it exists in an equilibrium between a folded state and an unfolded ensemble. Labeling SH3 with a fluorine on its sole tryptophan facilitates NMR-based detection of both states simultaneously, allowing quantification of the free energy of unfolding in vitro and in living E. coli cells. We find that hyperosmotic shock decreases SH3 stability, consistent with the idea that weak interactions are important under physiologically relevant crowded conditions. Subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.