10 Jun 2022
10 Jun 2022
Status: this preprint is currently under review for the journal MR.

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine

Sreelakshmi Mekkattu Tharayil1, Mithun C. Mahawaththa2, Akiva Feintuch3, Ansis Maleckis4, Sven Ullrich1, Richard Morewood1, Thomas Huber1, Christoph Nitsche1, Daniella Goldfarb3, and Gottfried Otting2 Sreelakshmi Mekkattu Tharayil et al.
  • 1Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
  • 2ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
  • 3Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
  • 4Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia

Abstract. The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag that reacts selectively with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between lanthanoid ion and protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP), (ii) reaction of the cyano groups with α-cysteine, penicillamine or β-cysteine to complete the lanthanide chelating moiety and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-amino thiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions.

Sreelakshmi Mekkattu Tharayil et al.

Status: open (until 08 Jul 2022)

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Sreelakshmi Mekkattu Tharayil et al.

Sreelakshmi Mekkattu Tharayil et al.


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Short summary
Having shown that tagging a protein at a single site with different lanthanoid complexes delivers outstanding structural information at a selected site of a protein (such as active sites and ligand binding sites), we now present a simple way by which different lanthanoid complexes can be assembled on a highly solvent-exposed cysteine residue. Furthermore, the chemical assembly is selective for selenocysteine, if a selenocysteine residue can be introduced into the protein of interest.