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

Abstract

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln III tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues.Loaded with Tb III or Tm III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

Localising nuclear spins by pseudocontact shifts from a single tagging site

“…Nonetheless, the amide protons with perpendicular metrics > 0.025 generally were associated with localisation spaces that closely trace the crystal structure (Figure 8). (Graham et al, 2011), whereas the C12 and C13 tags produce a thioether bond by nucleophilic substitution of the nitro group (Herath et al, 2021).…”

“…In the case of the ubiquitin data used in this work, the uncertainty of the PCS data associated with the amide protons of the segments A-C was small (estimated to be less than +/-0.01 ppm). A straightforward approach that indirectly captures the effect of most of the sources of uncertainty on the localisation spaces relies on performing the ∆χ-tensor fits multiple times with random omission of a certain percentage of the PCSs (Herath et al, 2021).…”

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine

“…A mutant aminoacyl-tRNA synthetase has been shown to install photocaged selenocysteine in proteins in response to an amber stop codon, providing a generally applicable route to genetic encoding of selenocysteine (Welegedara et al, 2018). Only few lanthanoid binding tags are suitable for forming stable ligation products with selenocysteine (Wu et al, 2017;Herath et al, 2021). Work is in progress to increase the protein yields with photocaged selenocysteine, as this would open a most attractive and widely applicable route to site-specific tagging of proteins with a short tether.…”

Rigid, Highly Reactive and Stable DOTA‐like Tags Containing a Thiol‐Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis**

“…Since a large number of DOTA-like tags and their protein conjugates have been reported, a comparison of the magnetic anisotropy generated by the reported DOTA-like tags and T1/ T2-Ln was made for the determined Δχ-tensor parameters [26,[35][36][37][38][39][40][41][42][43] and RDCs, [21,[36][37][38]40,[44][45][46][47] respectively. The chemical structures of the published tags [21,26,[36][37][38]40,[43][44][45][46][47] were shown in Figure S30, and the Δχ ax and the RDC determined alignment [a] All RDCs of backbone amides were measured at 298 K with a proton frequency of 600 MHz.…”