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

Angew Chem Int Ed Engl. 2020 Jun 22;59(26):10411-10415. doi: 10.1002/anie.202002288. Epub 2020 Apr 16.


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 1 H-15 N NMR spectroscopy, but is often limited by low signal-to-noise at pH>7 and T>293 K. We have developed an improved 13 Cα-13 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.

Keywords: NMR spectroscopy; cell signaling; kinases; phosphorylation; protein modifications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • BRCA2 Protein / chemistry
  • BRCA2 Protein / metabolism*
  • Carbon-13 Magnetic Resonance Spectroscopy
  • Humans
  • Hydrogen-Ion Concentration
  • Mitogen-Activated Protein Kinases / metabolism*
  • Nuclear Magnetic Resonance, Biomolecular
  • Octamer Transcription Factor-3 / chemistry
  • Octamer Transcription Factor-3 / metabolism*
  • Phosphorylation
  • Proto-Oncogene Proteins c-mdm2 / chemistry
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Temperature


  • BRCA2 Protein
  • BRCA2 protein, human
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Mitogen-Activated Protein Kinases