Screening protein-ligand interactions intracellularly using FRET hybrids

Protein-protein interactions are often mediated by specific protein modules that recognize a short linear peptide within a target protein. The ability to detect, identify, and modulate protein interactions inside living cells has, thus far, relied heavily upon transcriptional reporter-based two-hybrid or mass spectrometry approaches. We developed a novel quantitative methodology to identify protein-ligand interactions for known targets and iteratively optimize their properties in a broad range of cell types using Forster resonance energy transfer (FRET) between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Esherichia coli . Two types of widely occurring protein-interaction modules: the SH3C domain from Mona and the second PDZ (PDZ2) domain from PSD95 were used as target receptors. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly using a dilution series of cell lysates containing FRET hybrids, providing a new high-throughput approach to rank the affinity of many interaction partners.;To apply this approach in eukaryotic cells, the FRET hybrids system was further extended to yeast Saccharomyces cerevisiae. A random peptide library was screened for ligands of the PDZ2 domain. Randomly selected clones exhibited a clear consensus for the C-terminal PDZ binding motif -STXV-COOH with improved affinities when compared to those obtained in E. coli . Intracellular molecular evolution was performed by constructing a secondary focused library using the -STXV-COOH motif identified from the naive library. Screening of the focused library yielded a family of ligands with an extended consensus motif -[S/C][I/L]STXV-COOH and improvements in metrics used for screening (FRET signal and cell fluorescence intensity). Collectively, these results indicate that the use of FRET-hybrids in yeast provides an attractive opportunity for the development of affinity reagents for research and lead compounds for therapeutic development.