A protein mutation deemed innocuous under normal biochemical assay conditions can be destabilizing in the crowded environment of real cells, reports a team led by Gary J. Pielak of the University of North Carolina, Chapel Hill (Proc. Natl. Acad. Sci. USA 2015, DOI: 10.1073/pnas.1417415112). Researchers typically probe biomolecules suspended in dilute buffer, but a growing body of evidence suggests that proteins can behave differently in the cellular milieu, where concentrations of macromolecules can reach 300–400 g/L. Pielak and colleagues studied mutations of surface residues in a domain of protein G, a bacterial protein that binds antibodies, and compared the effects in buffer to those in living Escherichia coli cells. They tracked the stability of the protein by monitoring the exchange between hydrogen and deuterium in the protein backbone’s amide groups. Switching one amino acid from isoleucine to leucine didn’t have much effect. But a mutation of another residue from aspartate to lysine destabilized the protein in cells, even though the mutation appeared harmless in buffer. The results suggest that surface mutations as well as posttranslational modifications need to be studied under physiologically relevant conditions, the authors say.