Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Abstract

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones