Using NMR-detected backbone amide 1H exchange to assess macromolecular crowding effects on globular-protein stability.

The biophysical properties of proteins in the crowded intracellular environment are expected to differ from those for proteins in dilute solution. Crowding can be studied in vitro through addition of polymers at high concentrations. NMR-detected amide (1)H exchange is the only technique that provides equilibrium stability data for proteins on a per-residue basis under crowded conditions. We describe the theory behind amide (1)H exchange and provide a detailed description of the experiments used to quantify globular protein stability at the residue level under crowded conditions. We also discuss the detection of weak interactions between the test protein and the crowding molecules.