Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR
Résumé
In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.
Mots clés
Cell signaling
IDP
Intrinsically disordered proteins
Kinases
Multiple phosphosites
NMR spectroscopy
Phosphorylation
Post-translational modifications
Quantitative NMR
Time-resolved NMR
IDP phosphorylation monitored by NMR NMR spectroscopy
Intrinsically Disordered Proteins
post-translational modifications
phosphorylation
cell signaling
kinases
multiple phosphosites
quantitative NMR
time-resolved NMR
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