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

Abundant phosphorylation events control 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 H- N NMR spectroscopy, but is often limited by low signal-to-noise at pH>7 and T>293 K. We have developed an improved Cα- CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic-resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.