A chiral lanthanide tag for stable and rigid attachment to single cysteine residues in proteins for NMR, EPR and time-resolved luminescence studies

Iresha D Herath, Colum Breen, Sarah H Hewitt, Thomas R Berki, Ahmad F Kassir, Charlotte Dodson, Martyna Judd, Shereen Jabar, Nicholas Cox, Gottfried Otting, Stephen J Butler

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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 (NMR), electron paramagnetic resonance (EPR) and time-resolved luminescence spectroscopy experiments. Here we present a new stable chiral Ln(III) tag, referred to as C12, for spontaneous and quantitative reaction with a cysteine residue generating 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 (DEER) 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 timeresolved FRET assays and enzyme reaction monitoring.
Original languageEnglish
JournalChemistry - A European Journal
Publication statusAcceptance date - 9 Jun 2021

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