Protein Stability in Living Cells

Abstract

Theories concerning the effects of macromolecular crowding assert that the biophysical properties of proteins and nucleic acids can be significantly altered in native cellular environments relative to buffer alone. Despite a growing number of studies probing equilibrium thermodynamic protein stability in cells, there remains a lack of quantitative information, especially regarding residue-level stability under non-perturbing conditions. We have measured the in-cell stability of the 56-amino acid B1 domain of protein G (GB1) at the residue level without using destabilizing solutes or thermal modification by using NMR-detected hydrogen-deuterium exchange of quenched cell lysates. Comparison to dilute solution (pH 7.6 and 37 °C) shows that residues are stabilized in Escherichia coli cells by as much as 1.1 ±0.1 kcal/mol (Figure 1). We have also identified the residues most important for global folding of GB1 in cells. We discuss the implications of our findings with respect to structural models gleaned from studies conducted in buffer alone.View Large Image | View Hi-Res Image | Download PowerPoint Slide