Title: Seeing through protein complexes by high-throughput FRET
Abstract: Although fluorescence resonance energy transfer (also known as Förster-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 advances, FRET was regarded as a research method for computer and engineering geeks until the 1990s. The introduction of fluorescent monoclonal antibodies, green fluorescent protein derivatives, the development of FRET applications 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 excitation energy to the acceptor manifested in, among others, quenching of donor fluorescence and enhancement, or sensitization of acceptor fluorescence (7). FRET efficiency is supposed to be dependent only on the donor–acceptor distance, a property one expects from a “spectroscopic ruler.” Although in most cases this approximation is acceptable, the validity of the underlying assumption of dynamic averaging 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 literature abounds with methods using FRET intensity (fluorescence measured in the FRET channel) or uncalibrated FRET parameters. It is highly advisable to use the calibrated FRET efficiency instead of enigmatic and dubious parameters to prevent 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 membrane receptor complexes in intact cells (14). FRET is often used as a read-out parameter in assays in which the conformation 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 gaining more importance. Techniques used for the realization of high-throughput mapping of protein interactions (e.g., yeast two-hybrid, fluorescence complementation) are now supplemented by FRET-based sorting of cells in which certain proteins associate with each other (10, 16). In the current issue of Cytometry, Paar et al. (17) describe a high-throughput FRET method based on total internal reflection fluorescence microscopy (TIRFM). In TIRFM, the excitation light beam is reflected from the glass-sample interface back to the glass. Commonsense predicts that the light beam does not even reach the cells sitting on the coverslip. However, the incident beam generates an evanescent electromagnetic field penetrating into the cells. Since the evanescent field decays according to a steep exponential function, only fluorophores located within a couple of hundred nanometers from the interface will actually be excited. This circumstance limits the sensitive area of the microscope to a thin layer along the coverslip, therefore membrane proteins and transport vesicles close to the plasma membrane can selectively be investigated (18). The authors point out that ∼40% of the fluorescence intensity, which seems to be membrane-derived, is actually cytosolic in origin in confocal microscopy. This “contamination” of the membrane signal can be reduced to ∼15% by TIRFM. FRET and TIRFM have been combined earlier and this approach has been successfully used to monitor the conformational changes of G protein-coupled potassium channels upon activation (19) and to reveal the dimerization state of metabotropic glutamate receptor 1α (20). Paar et al. refurbished the combined FRET/TIRFM method and applied it to high throughput, high resolution imaging of large areas based on synchronizing sample movement and readout (17). They claim that throughputs of up to 1000 cells/min are achievable, and demonstrate that FRET can be accurately and reproducibly measured with their scanning system. After the determination of protein sequences and three-dimensional structures, there is a substantial need for the elucidation of protein interactions. Proteins carry out most of the activities, which turn a membrane-bound sack packed with organic compounds into a living cell, and they do so by interacting with each other in dynamic protein complexes. Elucidation of these interactions is not only required for an understanding of basic biological principles, but also for the development of selective medications. The combination of high-throughput approaches with quantitative analysis will turn FRET into an indispensable tool for the investigation of protein interactions. In the post-genomic era, the emerging fields of high content and high-throughput cell analysis are becoming more and more important. The majority of cell-based high-throughput assays are based on flow cytometry (16, 21-26), because this technique inherently provides a large amount of data within a short time; several parameters (up to 14) per cells or beads and several thousand cells or beads per second can be analyzed. Only a few image-based platforms have been described so far for high-throughput cell analysis (27-29), although the image-based approaches can reveal a different level of complexity of cellular systems. The FRET/TIRFM assay described by Paar et al. (17) is not simply just another high-throughput platform, but a significant advancement in monitoring membrane protein interactions in live cells.
Publication Year: 2008
Publication Date: 2008-03-13
Language: en
Type: letter
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 6
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