This proposal describes the design, synthesis and evaluation of several new fluorescent probes to be tested as membrane-permeant, selective epitope tags, compatible with living cells. These agents have been designed to form high affinity complexes with a short epitope sequences that contain four cysteine residues which are located in pairs separated by two turns of an alpha-helix. Means for optimizing fluorophore-epitope binding affinity and selectivity are described, including the preparation of one-bead one-compound epitope libraries, and phage display techniques. Finally, experiments are described to demonstrate """"""""proof of concept"""""""" by visualizing fluorescence resonance energy transfer within living cells, using the above-mentioned probes and recombinant proteins that incorporate suitable epitope sequences. Given the broad implications of protein-protein binding in nature, the availability of this method could provide an important new tool for the non-invasive study of numerous biological processes.
