A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR,EPR and Time-Resolved Luminescence Studies |
| |
Authors: | Iresha D. Herath Dr. Colum Breen Dr. Sarah H. Hewitt Dr. Thomas R. Berki Dr. Ahmad F. Kassir Dr. Charlotte Dodson Martyna Judd Dr. Shereen Jabar Dr. Nicholas Cox Prof. Gottfried Otting Dr. Stephen J. Butler |
| |
Affiliation: | 1. Research School of Chemistry, The Australian National University, Canberra, ACT 2605 Australia;2. Department of Chemistry, Loughborough University, Epinal Way, Loughborough, LE11 3TU UK;3. Department of Pharmacy & Pharmacology, University of Bath Claverton Down, Bath, BA2 7AY UK |
| |
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 LnIII 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 TbIII or TmIII 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 TbIII 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. |
| |
Keywords: | EPR spectroscopy FRET lanthanoid tags luminescence NMR spectroscopy |
|
|