Expression of fluorinated proteins in human cells for 19F in-cell NMR

In-cell NMR spectroscopy is a unique approach to study the structure and function of biological macromolecules in their native cellular environment at atomic resolution.1 At CERM/CIRMMP, we developed an approach for expressing and labelling proteins directly in human cells, which is ideally applied to monitor functional processes such as protein folding and maturation, metal binding, chemical modifications, and interactions with ligands or with specific partners.2 Such approach provides important insights on protein-ligand interactions, which are critical to optimize drug penetrance and potency.3 However, protein-observe drug screening by 1H and 1H-15N spectra is limited by fact that signals from proteins interacting with cellular components are broadened beyond detection. 19F NMR spectroscopy is an attractive alternative, thanks to the high-sensitivity and background-free nature of 19F. We have shown that fluorinated amino acids incorporated in proteins expressed in human cells allow protein-observed screening on otherwise invisible targets.4 Furthermore, the incorporation efficiency of fluorinated amino acids can be controlled by changing the media composition, and tuned to either maximise 19F signal intensity or to minimize structural perturbations.5 This approach holds great potential to study “invisible” protein targets in cells.