| Summary: | <p>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 (<span class="inline-formula">Δ<i>χ</i>)</span> 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 <span class="inline-formula">Δ<i>χ</i></span> 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.</p>
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