Intracellular protein interaction mapping with FRET hybrids.
A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using 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 Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.
You
Xia
X
Department of Chemical Engineering, University of California-Santa Barbara, Santa Barbara, CA 93106, USA.
Nguyen
Annalee W
AW
Jabaiah
Abeer
A
Sheff
Mark A
MA
Thorn
Kurt S
KS
Daugherty
Patrick S
PS
D002460:Cell Line; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins