title: FRETex: a FRET-based, high-throughput technique to analyze protein-protein interactions.
abstract: Protein-protein interactions (PPIs) are essential for cellular viability and activity. Here, we present a rapid, semi-quantitative method (termed FRETex) to analyze PPIs, taking advantage of the strong and specific FRET signal between fused CyPET donor and YPET acceptor molecules. To demonstrate the robustness of this approach, we analyzed the interactions between three protein pairs and their muteins: TEM1-β-lactamase binding its inhibitor BLIP, barnase binding barstar and ornithine decarboxylase binding its inhibitor antizyme. The CyPET/YPET fused proteins were produced in small quantities, and the measurements were conducted directly in the proteins crude Escherichia coli lysates without any purification step. Protein concentrations were determined from the fluorescence intensities of the lysates. While binding titration curves were produced, the resulting affinities were not always precise. Therefore, we also conducted time-resolved chase experiments using non-labeled binding partners as chasers. The acquired dissociation rate constants were in a good agreement with those measured by surface plasmon resonance. Due to the simplicity of FRETex, and the ability to obtain semi-quantitative binding data, FRETex is a suitable method for tasks such as mutant scans, protein-engineering, scanning for inhibitors and more.
authors.affiliation: Department of Biological Chemistry,  Weizmann Institute of Science, Rehovot 76100,  Israel.
authors.forename: Ruth
authors.initials: R
authors.lastname: Khait
authors.forename: Gideon
authors.initials: G
authors.lastname: Schreiber
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000191:economics / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D011485:Protein Binding; D015202:Protein Engineering; D025941:Protein Interaction Mapping / Q000191:economics / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D065108:Ornithine Decarboxylase Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D065093:beta-Lactamase Inhibitors; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D001618:beta-Lactamases; D009955:Ornithine Decarboxylase