Direct Protein Expression in Human Cells for the Investigation by In-cell NMR

In-cell NMR allows the studies of biomolecules in their native cellular environment. The information obtained from in-cell NMR studies will provide insights into the behaviours and states of the studied molecules. In this thesis, a brief overview of in-cell NMR, 19F in-cell NMR, mass spectrometry (MS) of intact proteins and the strategy of endogenous labelling of proteins in human cells are described. With the main focus on applying the direct expression of labelled proteins in human cells for in-cell NMR, three main research activities were investigated: (i) direct expression of fluorinated proteins in human cells for 19F in-cell NMR, (ii) controlling protein fluorination efficiency in human cells, and (iii) direct expression of SARS-CoV-2 proteins in human cells and their observations by in-cell NMR. The work established a new protocol for biosynthetic incorporation of fluorinated amino acids (FAA) into proteins in human cells, hence allowing the observations of proteins, protein-protein and -ligand interactions by 19F NMR. In addition, the experimental conditions to control the FAA incorporation probability in human cells were established by MS and NMR investigations. Lastly, the preliminary insights into SARS-CoV-2 proteins by in-cell NMR were obtained by both protein- and ligand-probed NMR experiments on live cells and cell lysates. These achievements resulted from an integration of different in-cell NMR approaches (19F NMR, 1H-15N HMQC and 1H-15N BEST-TROSY) and the allied techniques in structural biology, mass spectrometry and X-ray crystallography.