Understanding of cellular processes requires all the involved partners to be characterized at the structural level. In-cell NMR spectroscopy is a unique tool for characterizing bio¬logical macromolecules in the physiological environment of the living cell at atomic resolution. At CERM, we have devel¬oped a direct protein expression approach to observe proteins in human cells by NMR. This approach allows monitoring protein functional proc¬esses, such as protein folding and maturation, cofactor binding and redox changes in response to external factors, in their native environment.1

This approach was recently applied to investigate the interaction between an intracellular target protein and potential drugs in human cells. Such intracellular ligand screening approach could greatly benefit the early steps of drug development, as it allows both the structural characterization of the protein-ligand complex and the assessment of membrane permeability and binding selectivity towards the target, that are critical parameters to improve the potency of potential drugs.2

Finally, an advanced NMR bioreactor design is being implemented at CERM that greatly extends the lifetime of the cells up to several days. With this setup, protein conformational changes can be monitored in real time, providing a unique way to the study of complex kinetic behaviors of macromolecules in a cellular setting.3

(1) Luchinat, E.; Banci, L. In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation. Acc. Chem. Res. 2018, 51 (6), 1550–1557. https://doi.org/10.1021/acs.accounts.8b00147
(2) Luchinat, E.; Barbieri, L.; Cremonini, M.; Nocentini, A.; Supuran, C. T.; Banci, L. Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency. Angew. Chem. Int. Ed Engl. 2020. https://doi.org/10.1002/anie.201913436
(3) Cerofolini, L.; Giuntini, S.; Barbieri, L.; Pennestri, M.; Codina, A.; Fragai, M.; Banci, L.; Luchinat, E.; Ravera, E. Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions. Biophys. J. 2019, 116 (2), 239–247. https://doi.org/10.1016/j.bpj.2018.11.3132