The application of Förster Resonance Energy Transfer (FRET) has provided a new tool to study protein-protein interactions in both extra and intracellular environments, since the energy transfer between a donor and an acceptor is highly dependent on their distance (normally 1-10nm). Here, we present a novel methodology to discover putative protein interaction partners in the cytoplasm of Esherichia coli using high-throughput flow cytometry. Three general protein interaction modules were chosen as target proteins including one SH3 domain and two PDZ domains. Binding peptides containing physiologically relevant consensus sequences were identified and their binding affinities were quantitatively measured using FRET hybrids. This high-throughput affinity measurement method is unique among other protein-interaction screening assays since the binding events are dynamic and reversible. Also, we monitored protein-ligand interactions in the intracellular environment of eukaryotic cells including Saccharomyces cerevisiae and human embryonic kidney cells (293T) with improved FRET signals that suggest single step library screening could be achieved using flow cytometry, and peptide libraries were screened for PDZ domain ligands in Yeast. This novel methodology provides an effective means to map intracellular protein interactions and present opportunities to discover therapeutically useful protein interaction inhibitors.

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