Intracellular protein interaction mapping with FRET hybrids

Abstract

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.cell sorting ͉ protein-ligand interactions T he ability to identify and quantitatively characterize proteinprotein interactions in living cells is essential for developing a detailed, system-level understanding of cellular function. The yeast two-hybrid (Y2H) system has served as the primary genetic tool to discover potential biological interaction partners for an enormous number of proteins (1, 2). Application of Y2H assays to each of the nearly 6,000 yeast ORFs enabled construction of the first large-scale protein interaction network in yeast (3, 4), and similar interaction studies were performed subsequently in Drosophila melanogaster and Caenorhabditis elegans (5, 6). Human-protein interaction networks are less well characterized and present difficult challenges to interaction mapping approaches. In fact, only a small fraction of an estimated 10 5 to 10 6 human-proteome interactions (7) have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches (7,8). Unfortunately, high-throughput Y2H assays are typically prone to a high frequency of false positives that complicate the interpretation of interaction data. In some studies, it has been estimated that Ͼ50% of putative interactions identified are false positives (9).Several important challenges remain in the elucidation of protein-protein interaction networks and their influence on cell function (10). Large-scale interaction maps can portray basic network structure but lack quantitative features needed to understand network function. A predictive understanding of network function likely will require the elucidation of equilibrium and kinetic properties within a network, including individual equilibrium-binding constants. Furthermore, existing highthroughput approaches (e.g., Y2H) are not well suited to investigate real-time dynamics in protein networks essential for understanding cell function (11) because detection, in these cases, is ba...

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

“…We have previously shown that this technique can be used to detect molecular interactions in situ in Drosophila brain tissue (26). FRET has been previously shown to identify proteins that are in extremely close proximity (Ͻ5 nm) and is highly correlated with a direct molecular interaction (27). Drosophila brain tissues were incubated with antibodies directed against Polo kinase and RacGAP or Pav-KLP and stained with secondary antibodies labeled with the Cy3 and Cy5 fluorophores, respectively.…”

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

“…To address this shortcoming, several groups have exploited oligomerizationassisted reassembly of split enzymes such as adenylate cyclase (20), ␤-lactamase (Bla) (21), and dihydrofolate reductase (22,23), as well as split fluorescent proteins (24,25). Alternatively, a number of methodologies for detecting interacting proteins in bacteria have been developed that do not rely on interaction-induced complementation of protein fragments, but instead use phage display (26), FRET (27), and cytolocalization of GFP (28).…”

Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease

“…These reporters consist of an optimized FRET pair of fluorescent proteins, YPet and CyPet, which were recently described by Daugherty and coworkers [24] for the study of molecular interactions in vivo [39]. The CyPet and YPet proteins were linked together by a flexible linker containing a consensus recognition site for LF protease [36] flanked at both its N-and C-termini by several repeats of the flexible tripeptide Gly-Gly-Ser [41].…”