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. 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 in E.coli 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. High throughput screening for protein binding ligands was also extended to Saccharomyces cerevisiae, demonstrating that high affinity peptides could be identified using fewer rounds of enrichment in eukaryotic cells due to its significantly increased dynamic range comparing to previous studies.

1. Stathakis, D. G., Hoover, K. B., You, Z. & Bryant, P. J. (1997) Genomics 44, 71-82. 2. Bourette, R. P., Arnaud, S., Myles, G. M., Blanchet, J. P., Rohrschneider, L. R. & Mouchiroud, G. (1998) Embo J 17, 7273-81.