The conformation and association state of proteins are recognized as important factors in biological regulation. Fluorescence resonance energy transfer (FRET) is a physical phenomenon that can be used to measure in a quantitative way the average separation distance between two molecules (1). Although it was described in the 1940s by Dexter (2) and Förster (3), it has become a widely accepted biological research tool only in the past decade. In FRET, energy is transferred in a non-radiative fashion among an excited fluorescent molecule, the donor, and a nearby acceptor molecule that is usually, but not necessarily, fluorescent (4). Energy transfer efficiency (E) is defined as the fraction of excited donor molecules undergoing FRET and can be expressed in the following way:
where R is the separation distance between the donor and the acceptor and Ro is a distance at which E is 50%. Ro is characteristic of a given donor-acceptor pair and its value is typically in the 3- to 8-nm range. The rate of FRET efficiency depends on the sixth power of the separation distance between the donor and the acceptor, thus providing a sensitive tool for the measurement of protein associations in the 2- to 10-nm range.
Before the introduction of green fluorescent protein (GFP) and its variants, which we collectively refer to as visible fluorescent proteins (VFPs), FRET measurements were limited to intrinsically fluorescent proteins or to proteins labeled with fluorescent ligands or antibodies. The advent of VFP-tagged proteins made FRET measurements in unperturbed systems much more widely applicable. Cyan and yellow fluorescent proteins (CFP-YFP) and blue fluorescent protein (BFP) and GFP are good donor-acceptor pairs based on their Ro values and the overlap between donor and acceptor excitation and emission wavelengths (5). Many methods are available for the measurement of FRET. Several recent papers have been devoted for a comprehensive review of the topic (6–8), so we only briefly refer to flow cytometrically based FRET methods and to problems related to reliable quantification of FRET experiments.
In many cases, investigators have calculated FRET intensities or indices instead of reporting FRET efficiencies. FRET intensity is the fluorescence intensity measured in the FRET channel after correction for spectral overspill (9). It is sensitive for the amount of fluorophores expressed and thus is very error-prone. FRET indices are functions of the FRET efficiency only, but their interpretation is made difficult by the absence of a clear physical meaning of the calculated parameter (10). In addition, some of them may show interlaboratory variation. FRETN (11) and N-FRET (12) are FRET indices that take the donor and acceptor concentrations into account and are supposed to be true measurements of protein-protein interactions (11, 12). Lately, their utility has been questioned based on their non-monotonous dependence on the acceptor concentration (13). Although methods based on the detection of increased donor anisotropy in the presence of FRET have a solid physical background, their results are difficult to interpret in terms of protein associations without resorting to complicated modeling (14–16).
A plethora of approaches exists that yields the FRET efficiency itself. We previously reported one such method based on the combined detection of donor and acceptor fluorescence intensities in cells labeled with fluorescent ligands or antibodies using flow cytometry (17, 18). Later, we established a similar method in fluorescence microscopy (19). These methods require a factor, variably called α (17–19) or G (11, 13), which relates the intensity lost on the donor side due to donor quenching to the enhanced emission of the acceptor due to sensitized emission. Methods for the determination of α have been reported for antibody-labeled cells in flow cytometry (17, 18) and microscopy (19) and lately for VFP-expressing cells in microscopy (13). To the best of our knowledge, successful determination of α has not been reported for VFP-expressing cells using flow cytometry. A popular approach for the determination of FRET efficiency is based on the release of donor quenching upon photodestruction of the acceptor (20, 21). Although this method has been applied to conventional fluorophores (20) and VFP-expressing cells (21), it cannot be used in flow cytometry because detection of donor fluorescence intensity before and after acceptor photobleaching is usually not feasible.
Because flow cytometry is statistically superior to fluorescence microscopy, it is an attractive method for FRET measurements. As discussed above, accurate determination of the FRET efficiency in VFP-expressing cells has not been accomplished in flow cytometry. We present such an approach, and its novelty lies in the determination of α in cells expressing VFP using flow cytometry. We demonstrate the reliability of the approach using cells with and without interaction between the donor and acceptor VFP derivatives.
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We have developed a novel flow cytometric method for the quantitative evaluation of the FRET efficiency between different VFPs on a cell-by-cell basis. A flow cytometric FRET calculation must involve a constant, designated α in the present report, that relates the fluorescence intensity of acceptor molecules to that of an equal number of excited donor molecules. The major merit of the present study is the elaboration of a novel method for the flow cytometric determination of α. In microscopy the determination of α can be based on the comparison of the intensity lost on the acceptor side due to acceptor bleaching to the consequent increase in donor intensity due to release of quenching (13). Because photobleaching measurements are difficult to carry out in flow cytometry, the usual flow cytometric way to determine α is to compare the fluorescence intensity of an equal number of excited donor and acceptor molecules. When antibody- or ligand-labeled cells are used, this is usually done by separately labeling an equal number of epitopes with donor- and acceptor-tagged antibodies. Identical expression of two VFPs serving as a donor and acceptor pair can be achieved only if the two are part of a single protein. If there is no FRET between the donor and acceptor, α can be directly calculated using equation 18 provided the absorption ratio is known. We wanted to create such a CFP-YFP chimera by placing a linker consisting of 30 prolines between the two VFPs (31). If there had been no FRET between CFP and YFP in such a construct, it could have been used for the determination of α directly as described previously (17, 18). However, initial measurements showed that FRETN and N-FRET parameters were not 0, indicating that FRET took place. The lack of correlation between the FRET efficiency and the expression level of the fusion construct indicated that intermolecular FRET did not play a role. Taking into account that the Ro for the CFP-YFP pair is 4.9 nm (5), a FRET efficiency of 10% corresponds to a donor-acceptor separation distance of 7 nm. In a typical polyproline helix in an aqueous environment (polyproline helix type II), the helical rise per residue is 0.31 nm, yielding 9.3 nm for the separation between the N- and C-termini of the polyproline linker expected to result in a FRET efficiency of 2%. If the fluorophores are assumed to be at the end of the helix, the polyproline linker has to be significantly bent (with a curvature radius of 3.8 nm). Although polyproline helices can be distorted by asymmetric hydrogen bonding with surrounding water molecules, such a significant bending is unlikely. In contrast, CFP and YFP attached to the termini of the linker may swing toward each other, thus decreasing the separation distance between the fluorophores below the length of the linker. The presence of FRET in the CFP-30Pro-YFP sample was the major motive to elaborate the method for the determination of α described in this report, which does not rely on the lack of FRET in the calibration samples or knowledge of the absorption ratio.
Our method requires at least two samples with different FRET efficiencies expressing donor-acceptor chimeras strictly generating a donor to acceptor ratio of 1. The reliability of the method is significantly enhanced if there are at least three calibration samples. FRET is calculated using two different approaches (equations 10 and 14) that yield two FRET-related ratios (RI and RF). The relation between RI and RF is determined by the absorption ratio and α according to equations 16 and 17. These equations can be used to derive the absorption ratio and α in a single step by fitting equation 16 or 17 to the RF-RI or 1/(RF − 1) − 1/RI plot, respectively. From a practical point of view, the number of calibration samples is usually small; in our case it was three, so fitting two parameters to three measurement data points is prone to errors. If there were a much larger number of calibration samples, fitting of equation 16 or 17 could be reliably used, but the requirement for a larger set of calibration samples would make the method more costly and laborious. Our method eliminates this drawback by creating a ratio norm whose squared sum can be minimized by changning only α (equation 15) and then finding the absorption ratio by linear regression (equation 11). We verified our flow cytometric method by determining the FRET efficiency of the CFP-YFP construct containing a trypsin-sensitive linker by trypsin digestion, which induces the separation of the donor and the acceptor and a concomitant de-quenching of the donor (26). This measurement yielded a FRET value not significantly different from our flow cytometric determination.
The dependence of the MSE on α was relatively weak if α was large, especially above the minimum (equation 19, Fig. 3). However, the 95% confidence interval of α was not substantially different between small and large values of α due to presence of a tail in the frequency histogram of calculated α values above the optimal value of α if α was small (Fig. 4).
We have successfully applied the described approach for the CFP-YFP pair, but we could not achieve a reliable determination of α with the BFP-GFP donor-acceptor pair. The major cause of the failure must have been the low fluorescence quantum yield and high bleaching rate of BFP (32) and the high level of autofluorescence in this spectral region. Low donor intensity severely increases the error of equation 10 (18). In addition, equation 8 shows that a low donor fluorescence quantum yield increases α, resulting in a weak dependence of the MSE on α.
Finding interaction partners of a protein is a frequent problem in the post-genomic era. Currently, yeast two-hybrid (33, 34) and fluorescence complementation (35) techniques are used to find interaction partners for a protein of interest. Both approaches are based on the reconstitution of some kind of an activity (e.g., transcription, fluorescence) after two halves of a protein are brought together by the association of two interacting proteins the two fragments are genetically fused to. A lingering question about these methods is whether an interaction is driven by the two halves of the transcription factor (yeast two-hybrid) or VFP (fluorescence complementation) or by the proteins they are fused to. We demonstrated that cells can be sorted based on FRET. Sorting was done based on the intensity in the FRET channel, which is determined not only by FRET. Faster electronics can perform online FRET calculations according to equation 10, making sorting based on accurate FRET values possible. If a bait is fused to CFP and a library of YFP-fused proteins is generated, then FRET can be used as a marker for the interaction and cells can be sorted accordingly (36). Similarly to the split ubiquitin system (33), our approach is exquisitely suited for the study of membrane protein interactions, an area that was not amenable to yeast two-hybrid experiments.
The deficiency of calculating fluorescence intensities in the FRET channel instead of real FRET efficiencies is demonstrated by a recently published report in which the investigators examined the interaction between CFP- and YFP-tagged proteins in live yeast cells based on measuring FRET intensities (9). Due to the sensitivity of FRET intensity for the expression level of VFPs, the distinction between “significant” and “nonsignificant” FRET intensity is rather subjective, probably resulting in an underestimation of the fraction of cells showing FRET, as the investigators acknowledge. These investigators (9) also suggested using FRET measurements to screen VFP-tagged proteins for interactions. We believe that this could be accomplished using our quantitative approach in a much more sensitive way.
We have developed a method for the quantitation of FRET between VFPs in flow cytometry. Our approach eliminates the pitfalls of calculating FRET indices instead of FRET efficiency. We have demonstrated the successful application of the method for the CFP-YFP donor-acceptor pair. Quantitative FRET measurements have the potential to identify and characterize molecular interactions in their native environment more accurately than currently used molecular biological techniques.