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Magnetic Resonance An interactive open-access publication of the Groupement AMPERE
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© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.

  04 Nov 2020

04 Nov 2020

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This preprint is currently under review for the journal MR.

Phosphoserine for the generation of lanthanide binding sites on proteins for paramagnetic NMR

Sreelakshmi Mekkattu Tharayil1,, Mithun Chamikara Mahawaththa1,, Choy-Theng Loh1,a, Ibidolapo Adekoya1, and Gottfried Otting1 Sreelakshmi Mekkattu Tharayil et al.
  • 1ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra ACT 2601, Australia
  • apresent address: Hangzhou Wayland Bioscience Co. Ltd, Hangzhou 310030, PR China
  • These authors contributed equally to this work.

Abstract. Pseudocontact shifts (PCS) generated by paramagnetic lanthanide ions provide valuable long-range structural information in 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.

Sreelakshmi Mekkattu Tharayil et al.

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Sreelakshmi Mekkattu Tharayil et al.

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NMR spectra Sreelakshmi Mekkattu Tharayil, Mithun Chamikara Mahawaththa, Choy-Theng Loh, Ibidolapo Adekoya, and Gottfried Otting

Sreelakshmi Mekkattu Tharayil et al.


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