I will discuss the successes and challenges of in-cell protein NMR and the

quantification of protein stability under crowded conditions.  One

challenge is the inability to observe 15N-1H HSQC spectra from most

globular proteins in cells.  To understand this problem we turned to in

vitro experiments, using synthetic polymers, globular proteins, and cell

lysates to assess how these crowding agents affect protein diffusion.  To

examine stability, we used NMR-detected amide-proton exchange to quantify

the opening free energy of test proteins in the presence of crowding

agents and in Escherichia coli cells.  Our results highlight the

differences between crowding agents and shed light on the biological

effects of crowding.