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- Title
Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine.
- Authors
Tharayil, Sreelakshmi Mekkattu; Mahawaththa, Mithun C.; Feintuch, Akiva; Maleckis, Ansis; Ullrich, Sven; Morewood, Richard; Huber, Thomas; Nitsche, Christoph; Goldfarb, Daniella; Otting, Gottfried
- 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 bcysteine 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. addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with Gd3+ ions.
- Subjects
RARE earth metals; BINDING sites; PROTEIN expression; ELECTRONS; CYSTEINE
- Publication
Magnetic Resonance, 2022, p1
- ISSN
2699-0016
- Publication type
Article
- DOI
10.5194/mr-2022-11