Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy

Abstract

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.

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…179−181 A phosphoserine residue in conjunction with an aspartate or glutamate residue, or two phosphoserine residues together with a glutamate residue can generate Ln(III) binding sites that position the metal ion very precisely on the target protein, generating substantial Δχ tensors. 182 Unfortunately, the close proximity of charged amino acid side chains produces significant electrostatic repulsion, which can lead to unfolding of the protein and poor protein yields.…”

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time‐Resolved Luminescence Studies

“…Finally, while double‐arm tags attached to two neighbouring cysteine residues immobilise metal ions more easily,[ 81 , 86 , 87 , 88 , 89 , 90 ] suitable sites for double‐cysteine mutations require careful selection and stable attachment of the tag is not guaranteed. [91] Strategies that immobilize the lanthanide ion by simultaneous coordination to two phosphoserine residues [92] or chelating moieties installed on neighbouring cysteine residues[ 93 , 94 ] are similarly restricted in the choice of attachment sites. By allowing attachment to a single amino acid residue, the C12 tag opens a much greater choice of suitable tagging sites.…”

The evolution of paramagnetic NMR as a tool in structural biology

“…Lanthanoid binding tags rigidly attached to diamagnetic systems 59–62 or specific lanthanoid-binding sites introduced in diamagnetic proteins 63 are conveniently used for the generation of paramagnetic data to be used as structural restraints in the study of diamagnetic biomolecules. It was recently made clear that the choice of the lanthanoid ligand can significantly affect the magnitude of the magnetic susceptibility anisotropy, and thus of the PCSs, even in the assumption of a completely rigid attachment to the biomolecule.…”