Bacterial expression systems have been engineered to incorporate over 100 different unnatural amino acids into polypeptide chains in response to an amber stop codon (Liu and Schultz, 2010; Wan et al., 2014). The properties of some of these amino acids make them uniquely suited to serve as site-specific NMR “spies” in polypeptides, by providing an NMR signal that stands out against the background of the signals from the natural amino acid residues. This can be used to detect responses to ligand binding, measure binding affinities, attach paramagnetic tags, identify the location of the unnatural amino acid with respect to a protein and add chemical modifications in a biorthogonal manner. The presentation will demonstrate the advantage of cell-free systems for the incorporation of new and chemically sensitive unnatural amino acids for applications in structural biology (Loh et al., 2018; Loscha et al., 2014; Mahawaththa et al., 2018).

Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444.

Loh CT, Adams LA, Graham B, Otting G (2018) Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy. J. Biomol. NMR 71, 287–293.

Loscha KV, Herlt AJ, Qi R, Huber T, Ozawa K, Otting G (2012) Multiple-site labeling of proteins with unnatural amino acids. Angew. Chem. Int. Ed. 51, 2243–2246.

Mahawaththa MC, Lee MD, Giannoulis A, Adams LA, Feintuch A, Swarbrick JD, Graham B, Nitsche C, Goldfarb D, Otting G (2018) Small neutral Gd(III) tags for distance measurements in proteins by double electron-electron resonance experiments. Phys. Chem. Chem. Phys. 20, 23535–23545.

Wan W, Tharp JM, Liu WR (2014) Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochem. Biophys. Acta 1844, 1059–1070.