FRET is a powerful tool to study protein-protein interactions in living cells. Previous studies have mainly focused on intracellular sensing of known or putative protein interactions. We developed an efficient methodology to perform high throughput screening of libraries to discover novel protein binding ligands in the intracellular environment. Improved FRET pairs were genetically fused to target proteins and a large peptide library correspondingly. Binding brings the FRET donor and acceptor into close proximity, triggering resonance energy transfer and a spectrum shift upon donor excitation. Two well-conserved interaction modules: one PDZ domain from PSD95(1) and an SH3 domain from Mona(2) were fused to FRET acceptor as baits and a 15mer random peptide library were fused to FRET donor as a prey. Optical screening via flow cytometry revealed a set of physiologically relevant consensus motifs with improved binding affinities from both modules, demonstrating the potential application in large scale protein-interaction mapping and development of peptide inhibitors for intracellular targets. The FRET hybrids approach has been validated in both bacteria and yeast cells, and known protein interactions model was also established in mammalian cells with significantly increased dynamic range comparing to previous studies, implying this novel means can be extended to all types of cells.

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