posters 5th Asia-Pacific NMR Symposium 2013

Site-specific labelling of proteins with a lanthanide binding tag attached to an unnatural amino acid for the generation of pseudocontact shifts (#172)

Choy Theng Loh 1 , Bim Graham 2 , Gottfried Otting 1
  1. Research School of Chemistry, The Australian National University, Acton, ACT, Australia
  2. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia

Paramagnetic labels have emerged as a powerful tool for structural studies of proteins and protein-ligand complexes. In particular, pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions are easy to measure in NMR spectra and provide long-range structural information up to about 40 Å.12 To generate useful PCS data, however, the lanthanide tag must be attached to the protein site-specifically and with minimal mobility of the metal ion.

While most of the traditional lanthanide tags rely on single cysteine residues for site-specific attachment to proteins, a more generally applicable method employs unnatural amino acids. We used an orthogonal suppressor-tRNA/aminoacyl-tRNA synthetase pair developed by the group of P. G. Schultz for site-specific incorporation of p-azido-phenylalanine (AzF) into different target proteins.3 Subsequently, the AzF residue was reacted with a lanthanide-binding tag in a Cu(I)-catalyzed cycloaddition reaction.4 We show that sizeable PCSs can be generated in this way using lanthanide complexes of an alkyne derivative of nitrilotriacetic acid, which is a relatively small tag.

  1. Otting, G. (2008) Prospects for lanthanides in structural biology by NMR. J. Biomol. NMR 42, 1–9.
  2. Otting, G. (2010) Protein NMR using paramagnetic ions. Annu. Rev. Biophys. 39, 387–405.
  3. Wang, L. and Schultz, P. G. (2004) Expanding the genetic code. Angew. Chem. Int. Ed. 44, 34–66.
  4. Loh, C. T., Ozawa, K., Tuck, K. L., Barlow, N., Huber, T., Otting, G. and Graham, B. (2013) Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins. Bioconjugate Chem. 24, 260-268.