The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast,Selective and reversible Paramagnetic Tagging of Proteins |
| |
| Authors: | MSc Maxime Denis MSc Charlotte Softley Dr Stefano Giuntini Dr Matteo Gentili Dr Enrico Ravera Prof Giacomo Parigi Prof Marco Fragai Dr Grzegorz Popowicz Prof Michael Sattler Prof Claudio Luchinat Dr Linda Cerofolini Prof Cristina Nativi |
| |
| Institution: | 1. Giotto Biotech, S.R.L, Via Madonna del piano 6, 50019 Sesto Fiorentino (FI), Italy;2. Biomolecular NMR, Department Chemie, Technical University of Munich, Lichtenbergstrasse 4, 85747 Garching, Germany;3. Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy;4. Magnetic Resonance Center (CERM), University of Florence, and Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (C.I.R.M.M.P), Via L. Sacconi 6, 50019 Sesto FIorentino (FI), Italy;5. Institute of Structural Biology, Helmholtz Center Munich, Neuherberg, Germany |
| |
| Abstract: | 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. |
| |
| Keywords: | biomolecular NMR lanthanide coordination paramagnetic tags photocatalyzed thiol-ene reaction protein structure refinement |
|
|
