TY - JOUR
T1 - Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
AU - Mekkattu Tharayil, Sreelakshmi
AU - Mahawaththa, Mithun C.
AU - Feintuch, Akiva
AU - Maleckis, Ansis
AU - Ullrich, Sven
AU - Morewood, Richard
AU - Maxwell, Michael J.
AU - Huber, Thomas
AU - Nitsche, Christoph
AU - Goldfarb, Daniella
AU - Otting, Gottfried
N1 - Publisher Copyright:
© Author(s) 2022.
PY - 2022/9/13
Y1 - 2022/9/13
N2 - 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 for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the 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 lanthanoid 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-aminothiol 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.
AB - 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 for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the 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 lanthanoid 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-aminothiol 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.
UR - http://www.scopus.com/inward/record.url?scp=85140645754&partnerID=8YFLogxK
U2 - 10.5194/mr-3-169-2022
DO - 10.5194/mr-3-169-2022
M3 - Article
SN - 2699-0059
VL - 3
SP - 169
EP - 182
JO - Magnetic Resonance
JF - Magnetic Resonance
IS - 2
ER -