A flow cytometric method to detect protein-protein interaction in living cells by directly visualizing donor fluorophore quenching during CFP-->YFP fluorescence resonance energy transfer (FRET).
BACKGROUND
Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency.


METHODS
A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by comparing CFP intensity between FRET-positive and -negative populations. To accomplish this, we compared the CFP intensity in FRET-positive and FRET-negative cells within the same population transfected with a CFP/YFP fusion protein, in which the molar ratio of CFP:YFP was one. By using separate lasers to excite CFP and YFP, the detection of FRET was separated from that of YFP. Therefore, after direct excitation, the YFP emission spectrum remained constant in all transfected cells, whereas the emission spectrum of CFP varied with the extent of FRET in individual cells. Specific CFP/YFP fusion constructs were prepared to evaluate this approach. The first one consisted of CFP and YFP separated by two caspase cleavage sites (CFP-LEVD-YFP). A second construct consisted of CFP and YFP separated by a structurally restricted 232-amino acid (aa) spacer. No FRET was observed by transfectants expressing this construct.


RESULTS
Transfection of CFP-LEVD-YFP into Hela cells resulted in a FRET-positive population and a FRET-negative one. The appearance of the FRET-negative population was inhibited by the caspase inhibitor z-VAD. Moreover, substituting D for A in the caspase cleavage sites of this probe abolished the FRET-negative population, demonstrating the probe's specificity for caspase activity. Comparison of the CFP emission in the FRET-positive and FRET-negative population was used to document the relationship of FRET to donor quenching and permit the calculation of FRET efficiency and relative molecular distance between CFP and YFP. Similar results were noted when cells transfected with the caspase-sensitive probe (in the presence of z-VAD) were mixed with cells expressing the CFP-YFP construct with the 232-aa spacer and therefore were FRET negative. This demonstrated the validity of calculating CFP donor quenching and FRET efficiency by comparing emission spectra of an unknown construct with that of a known positive control, both expressed by the same population of cells. Using this approach, we confirmed that members of the TNF receptor-associated factor (TRAF) family engaged in both homotypic and heterotypic interactions.


CONCLUSIONS
We have established a novel flow cytometric approach to assess donor CFP quenching during CFP-->YFP FRET, which can be used for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties. This method can be used not only to analyze cells that express a CFP and YFP fusion protein, but also independent CFP-coupled and YFP-coupled interacting proteins.
D017209:Apoptosis / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048008:TNF Receptor-Associated Factor 3; D048015:TNF Receptor-Associated Factor 5
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D048008:TNF Receptor-Associated Factor 3; D048015:TNF Receptor-Associated Factor 5; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D020169:Caspases