Abstract. Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling
proteins site-specifically with a single lanthanide ion remains an ongoing
challenge, especially for proteins that are not suitable for ligation with
cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine
in conjunction with other negatively charged residues, such as aspartate,
glutamate or a second phosphoserine residue. The close proximity of the
binding sites to the protein backbone leads to good immobilization of the
lanthanide ion, as evidenced by the excellent quality of fits between
experimental PCSs and PCSs calculated with a single magnetic susceptibility
anisotropy (Δχ) tensor. An improved two-plasmid system was
designed to enhance the yields of proteins with genetically encoded
phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt
bridges, although the presence of too many negatively charged residues in
close proximity can also lead to unfolding of the protein. In view of the
quality of the Δχ tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine,
this method presents an attractive tool for generating PCSs in stable
proteins, particularly as it is independent of cysteine residues.
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 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.
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.
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