title: Residue level quantification of protein stability in living cells.
abstract: The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.
authors: Department of Chemistry.
authors: William B
authors: WB
authors: Monteith
authors: Department of Chemistry,Department of Biochemistry and Biophysics, andLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 gary_pielak@unc.edu.
authors: Gary J
authors: GJ
authors: Pielak
mesh_terms: D000577:Amides; D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D050296:Microbial Viability; D009587:Nitrogen Isotopes; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012996:Solutions; D013816:Thermodynamics
chemical_list: D000577:Amides; D000596:Amino Acids; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D009587:Nitrogen Isotopes; D012996:Solutions
keywords: H/D exchange; macromolecular crowding; protein NMR; protein thermodynamics