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

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

  • J Med Chem. 2023 Mar 15. doi: 10.1021/acs.jmedchem.3c00222.
Kasuni B Ekanayake 1,  Mithun C Mahawaththa 1,  Haocheng Qianzhu 2,  Elwy H Abdelkader 1,  Josemon George 2,  Sven Ullrich 2,  Rhys B Murphy 2,  Sarah E Fry 3 4,  Jason Johansen-Leete 3 4,  Richard J Payne 3 4,  Christoph Nitsche 2,  Thomas Huber 2,  Gottfried Otting 1
Affiliations

Affiliations

  • 1 Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Acton, Canberra, Australian Capital Territory 2601, Australia.
  • 2 Research School of Chemistry, Australian National University, Acton, Canberra 2601, Australia.
  • 3 Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, New South Wales 2006, Australia.
  • 4 School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
PMID: 36920850 DOI: 10.1021/acs.jmedchem.3c00222
Abstract

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.

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