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ACS Chem Biol. 2014 Nov 21;9(11):2502-7. doi: 10.1021/cb500658w. Epub 2014 Oct 6.

Designed phosphoprotein recognition in Escherichia coli.

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Department of Molecular Biophysics and Biochemistry, ‡Integrated Graduate Program in Physical and Engineering Biology, §Department of Chemistry, ∥Department of Cellular and Molecular Physiology, ⊥Systems Biology Institute, and #Department of Molecular, Cellular, and Developmental Biology, Yale University , New Haven, Connecticut 06520, United States.


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 Escherichia coli. 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 in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

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