Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine

• Protein stability is quantified in living cells using 19F NMR spectroscopy.
• Osmotic shock destabilizes an SH3 domain in living cells.
• Glycine betaine restores SH3 stability after osmotic shock.

Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. 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 protein stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living Escherichia coli cells before and after hyperosmotic shock in the presence and absence of the osmolyte, glycine betaine. The 7-kDa N-terminal src-homology 3 domain of Drosophila signal transduction protein drk is used as the test protein. We find that hyperosmotic shock decreases SH3 stability in cells, consistent with the idea that transient attractive interactions are important under physiologically relevant crowded conditions. The subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient attractive interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.

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