Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA: PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. H-1 and H-1-N-15 correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform N-15 labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.