Seeing Through Protein Complexes by High-Throughput FRET Peter Nagy, 1 J anos Sz€ oll} osi 1,2 *  Key terms FRET; flow and image cytometry; protein clustering and confor- mation ALTHOUGH fluorescence resonance energy transfer (also known as F€ orster-type resonance energy transfer, FRET) was described as a physical phenomenon in the middle of the 20th century (1), it was an unrecognized tool in biology till the 1970s, when Stryer coined the term ‘‘spectroscopic ruler’’ to describe the unique capability of FRET to be used as a distance measuring method (2). Despite these initial ad- vances, FRET was regarded as a research method for computer and engineering geeks until the 1990s. The intro- duction of fluorescent monoclonal antibodies, green fluores- cent protein derivatives, the development of FRET applica- tions for flow cytometry (3,4), digital imaging microscopy (5,6), and the wide-spread availability of fast computers and flexible evaluation softwares turned FRET into a fashionable technique. In FRET, a fluorescent donor interacts with an acceptor molecule which is separated by 2–10 nm from the donor. The interaction results in the transfer of the donor excita- tion energy to the acceptor manifested in, among others, quenching of donor fluorescence and enhancement, or sen- sitization of acceptor fluorescence (7). FRET efficiency is supposed to be dependent only on the donor–acceptor dis- tance, a property one expects from a ‘‘spectroscopic ruler.’’ Although in most cases this approximation is acceptable, the validity of the underlying assumption of dynamic aver- aging has to be verified, because if not fulfilled, FRET also correlates with the relative orientation of the donor and the acceptor (8). Early approaches of FRET directly measured the donor- sensitized emission of the acceptor by using special narrow band-pass filter sets to eliminate spectral overspill between the donor, FRET, and acceptor channels (9). Even today, the litera- ture abounds with methods using FRET intensity (fluores- cence measured in the FRET channel) or uncalibrated FRET parameters. It is highly advisable to use the calibrated FRET ef- ficiency instead of enigmatic and dubious parameters to pre- vent drawing false conclusions. As the development in this area accelerates, new methods have been described for the accurate calculation of FRET efficiency between GFP variants (10), to account for the presence of free donors and acceptors (11) and to describe the proximity relationship of more than two epitopes (12,13). The fact that FRET reports the distance between the donor- and the acceptor-tagged epitopes, i.e., the conformation of the protein, in real-time in living cells made it possible to solve the three-dimensional structure of mem- brane receptor complexes in intact cells (14). FRET is often used as a read-out parameter in assays in which the conforma- tion of a sensor is affected by a protease, ligand, or ion. The combination of three fluorophores in a single FRET-based sensor makes simultaneous measurement of two parameters possible (15). In the post-genomic era, the protein interactome is gain- ing more importance. Techniques used for the realization of high-throughput mapping of protein interactions (e.g., yeast two-hybrid, fluorescence complementation) are now supple- mented by FRET-based sorting of cells in which certain pro- 1 Department of Biophysics and Cell Biology, University of Debrecen, H-4010 Debrecen, Hungary 2 Cell Biophysics Research Group of the Hungarian Academy of Sciences, University of Debrecen, H-4010 Debrecen, Hungary Received 31 January 2008; Accepted 7 February 2008 *Correspondence to: J anos Sz€ oll} osi, Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, POB39, H-4012 Debrecen, Hungary. E-mail: szollo@dote.hu Published online 13 March 2008 in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/cyto.a.20554 © 2008 International Society for Advancement of Cytometry Commentary Cytometry Part A  73A: 388389, 2008