Journal
CHEMPHYSCHEM
Volume 21, Issue 9, Pages 863-869Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cphc.202000071
Keywords
biomolecular NMR; lanthanide coordination; paramagnetic tags; photocatalyzed thiol-ene reaction; protein structure refinement
Funding
- European Union [675555]
- University of Florence
- Recombinant Proteins JOYNLAB
- Regione Toscana (CERM-TT)
- Instruct-ERIC, a landmark ESFRI project
- CERM/CIRMMP Italy
- COST Action [CA15209]
- iNEXT - Horizon 2020 program of the European Union [653706]
- Regione Toscana (BioEnable)
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Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.
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