Sensitivity‐Enhanced 13C‐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 theoreticall...

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Bibliographic Details
Published in:Angewandte Chemie 2020-06, Vol.132 (26), p.10497-10501
Main Authors: Alik, Ania, Bouguechtouli, Chafiaa, Julien, Manon, Bermel, Wolfgang, Ghouil, Rania, Zinn‐Justin, Sophie, Theillet, Francois‐Xavier
Format: Article
Language:English
Subjects:
BRCA2 protein
Breast cancer
cell signaling
Chemistry
Deoxyribonucleic acid
DNA
DNA repair
Gene regulation
Kinases
Magnetic resonance spectroscopy
MDM2 protein
Monitoring
NMR
NMR spectroscopy
Nuclear magnetic resonance
Oct-4 protein
pH effects
Phosphorylation
Physiology
protein modifications
Proteins
Sensitivity enhancement
Spectroscopy
Spectrum analysis
Temperature
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Summary: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 1H‐15N NMR spectroscopy, but is often limited by low signal‐to‐noise at pH>7 and T>293 K. We have developed an improved 13Cα‐13CO 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. An NMR spectroscopy method has been developed to monitor site‐specific phosphorylation at high pH and temperature, both of which are necessary for kinase activity but have previously prevented the thorough characterization of disordered proteins. This approach uses sensitivity‐enhanced 13Cα‐13CO experiments and is demonstrated using Mdm2, BRCA2, and Oct4. Using this approach, many more phosphorylation cases can now beinvestigated using NMR spectroscopy.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.202002288