A genetically encoded fluorescent sensor for protein Arg phosphorylation dynamics in live cells

Abstract

Protein Arg phosphorylation is a crucial post-translational modification (PTM) for stress response, virulence, and protein quality control of Gram-positive bacteria. Although this PTM was first discovered in the 1970s, it is only recently that a protein Arg kinase (McsB) and a pArg phosphatase (YwlE) have been identified in Gram-positive bacteria. McsB marks misfolded protein by Arg phosphorylation, and the pArg tag is recognized by the bacterial proteasome for degradation, analogous to protein ubiquitination in eukaryotes. Recently, we reported a CHEF-based fluorescent probe for real-time monitoring of Arg kinase/phosphatase activities in vitro. However, the peptide-based probe was not applicable to live-cell studies. To overcome this limitation, we developed new genetically encoded FRET-based enzyme activity probes for studying Arg phosphorylation dynamics in live cells. This sensor should be invaluable in studying the regulatory mechanisms of Arg phosphorylation dynamics.