title: Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.
abstract: 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.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.
authors.forename: Samantha S
authors.initials: SS
authors.lastname: Stadmiller
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.
authors.forename: Annelise H
authors.initials: AH
authors.lastname: Gorensek-Benitez
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Guseman
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 101 Manning Drive, Chapel Hill, NC 27514, USA. Electronic address: gary_pielak@unc.edu.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Pielak
mesh_terms: D001622:Betaine / Q000494:pharmacology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009997:Osmotic Pressure / Q000187:drug effects*; D055550:Protein Stability; D018909:src Homology Domains
chemical_list: D029721:Drosophila Proteins; C080530:drk protein, Drosophila; D001622:Betaine
keywords: glycine betaine; osmolytes; osmotic shock; protein NMR; protein stability