Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be
installed in proteins by genetic encoding. In addition, we describe
a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK),
which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2
main protease (Mpro) as a model system with three different
ligands, we show that the 1H and 19F nuclei
of the solvent-exposed trimethylsilyl and CF3 groups produce
intense signals in the nuclear magnetic resonance (NMR) spectrum.
Their response to active-site ligands differed significantly when
positioned near rather than far from the active site. Conversely,
the NMR probes failed to confirm the previously reported binding site
of the ligand pelitinib, which was found to enhance the activity of
Mpro by promoting the formation of the enzymatically active
dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive
path for site-specific NMR analysis of ligand binding to large proteins
of limited stability and at low concentrations.