Sensitivity-Enhanced 13C-NMR for Monitoring Multisite Phosphorylation at Physiological Temperature and pH

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Alik, Ania | Bouguechtouli, Chafiaa | Julien, Manon | Bermel, Wolfgang | Ghouil, Rania | Zinn-Justin, Sophie | Theillet, Francois-Xavier

Edité par CCSD ; Wiley-VCH Verlag -

International audience. Abundant phosphorylation events rule the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue-specific level using 1 H- 15 N NMR spectroscopy, but is often limited by low signal-to-noise at pH\textgreater7 and T\textgreater293K. We developed an improved 13 Cα- 13 CO correlation NMR experiment that works equally at any pH or temperature, i.e. also at those where genuine kinase activities occur. This permits to obtain atomic-resolution information in physiological conditions down to 25 μM. We exemplified the interest of this approach by monitoring phosphorylation reactions, in presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2 and Oct4.

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