A Genetically Encoded Fluorescent Sensor for Protein Arginine Phosphorylation

Protein arginine phosphorylation (pArg) is an important but underexplored post-translational modification (PTM). In Gram-positive bacteria, pArg serves as a "degron" to guide damaged proteins for degradation by the ClpCP protease. Accordingly, enzymes that regulate pArg have been investigated as potential therapeutic targets against drug-resistant bacteria. Despite its importance, monitoring pArg dynamics remains technically challenging due to the chemical instability of pArg, with no methods available for live-cell studies. To address this, we developed a genetically encoded fluorescent sensor, FLAP (FLuorescent Arg Phosphorylation sensor), based on FRET to monitor the activity of the arginine kinase McsB and the pArg phosphatase YwlE. FLAP exhibited reversible, real-time FRET changes upon arginine phosphorylation and dephosphorylation in vitro. In an orthogonal E. coli system, FLAP successfully detected McsB-dependent pArg formation upon expression of active McsB. These results establish FLAP as a genetically encoded platform for studying pArg-writing and pArg-erasing enzymes in vitro and provide a proof of concept for live-cell detection of Arg phosphorylation.