Structural interpretation of fluorescence resonance--energy transfer measurements

Fluorescence resonance —energy transfer (FRET) has beenwidely used to determine distance information in macromolecularsystems. However, little has been written about methods forcombining FRET distances into coherent structural models. Iargue that the methods used so far are inappropriate. This paperdescribes an algorithm specifically tailored for finding structuresfrom FRET measurements. This algorithm finds structures whichfit the experimentally measured parameter, the efficiency ofenergy transfer, rather than derived distances. The algorithmwas implemented in Mathematica and applied to FRET distancesobtained for the contractile protein actin. The approach usedis applicable to other experimental techniques which measuredistances between a relatively small number of loci. Received on November 17, 1990; accepted on March 20, 1991     

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Effective lattice behavior of fluorescence energy transfer at lamellar macromolecular interfaces.

Fluorescence energy transfer between donors and acceptors confined to macromolecular interfaces is considered. In particular, we discuss two theoretical models for the ensemble-average fluorescence intensity decay of the donor when both fluorophores are incorporated into a planar (e.g., lamellar) interface. The first model is based on a continuous distribution of donor and acceptor molecules on a two-dimensional surface, whereas the other assumes a discrete distribution of fluorophores along the nodes of a two-dimensional square lattice. Results for the discrete model show that the fluorescence intensity kinetics of a donor depends strongly on the geometry of the molecular distribution (i.e., the lattice constant) and the photophysics of fluorophores (i.e., critical radius of the energy transfer). Furthermore, a "discrete molecular distribution" might manifest itself in the experimental data as an increase in the apparent dimensionality of the energy transfer with increasing acceptor concentration. Altogether, the experimental and theoretical underpinnings indicate the enormous potential of using fluorescence energy-transfer kinetics for revealing structural features of molecular ensembles (i.e., geometry, shape) based on a single experimental measurement. However, further understanding the effects of restricted geometries on the fluorescence energy transfer is required to take full advantage of this information. Basic theoretical considerations to that end are provided.     

Myosin structure. Proximity measurements by fluorescence energy transfer

The results of energy transfer experiments on the proximity of six sites on the globular head region of myosin are discussed. A large hydrophobic crevice has been detected on each myosin head which is sufficiently large to accommodate six aromatic rings simultaneously. In the crevice is located a thiol residue not involved in activation of myosin Ca²⁺ ATPase and a lysine residue which is specifically trinitrophenylated with 2, 4, 6-trinitrobenzenesulfonic acid. A second sulfhydryl whose modification activates the Ca²⁺ ATPase is located near the hydrophobic thiol site. The tryptophan whose fluorescence is enhanced by ATP binding is sufficiently close to the thiols and lysine residue to quantitatively transfer its energy to probes at these sites. The site of myosin ATPase has been tentatively located as being near the other five sites by energy transfer to or from synthetic chromophoric substrates. Implications of these results on the possibility of determining the location of the myosin light chain and actin binding sites are discussed.     

Non-linear least-squares methods applied to the analysis of fluorescence energy transfer measurements.

We describe a new method for calculating the efficiency of fluorescence energy transfer on labeled macromolecules using steady-state measurements. A single estimation of the efficiency value is obtained by a global analysis of all the measurement data sets (absorption, emission and excitation spectra) using non-linear least-squares. The method was tested on simulated and experimental data obtained from three simple model compounds: an equimolar mixture of tryptophan-tyrosine and two peptides, Trp-Tyr and Trp-Gly-Gly-Tyr, in which transfer efficiencies are respectively nearly 100% and 50%. The method was found to be reliable and provides methodological and quantitative advantages in regard to the sequential methods currently used.     

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Sz?llosi et al., Cytometry, 5:210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes. This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.     

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.     

Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.     

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.     

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.     

Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system.

The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.     

Single-molecule fluorescence resonance energy transfer

Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation.     

Distance distributions in native and random-coil troponin I from frequency-domain measurements of fluorescence energy transfer

We used time-dependent fluorescence energy transfer to determine the distribution of donor-to-acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (Trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (Cys 133). The time-dependent intensity decays of the donor were measured by the frequency-domain method from 10 to 320 MHz. The frequency response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to A distances, using an algorithm that accounts for the intrinsic multiexponential decay of the donor. In the native state the D–A distribution is characterized by an average distance of 23 Å and a half-width of 12 Å. Denaturation results in a modest increase in the average distance to 27 Å, and a dramatic increase in half-width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.     

Calculation on fluorescence resonance energy transfer on surfaces.

A general method for estimating fluorescence resonance energy transfer between distributions of donors and acceptors on surfaces is presented. Continued fraction approximants are obtained from equivalent power series expansions of the change in quantum yield in terms of the fluorescent lifetimes or the steady-state fluorescence. These approximants provide analytic equations for the analysis of energy transfer and error bounds for the approximants. Specific approximants are derived for five models of interest for membrane biochemistry: (a) an infinite plane, (b) parallel infinite planes, (c) the surface of a sphere, (d) the surfaces of concentric spheres, and (e) the surfaces of two separated spheres. Recent experimental results in the literature are analyzed with the equations obtained.     

Phospholipid vesicle fusion monitored by fluorescence energy transfer.

A method is presented which allows the observation of phospholipid vesicle fusion by the occurrence of Förster resonance energy transfer between the amphiphilic probes dansyldipalmitoylphosphatidylethanolamine and 3-[4-($\text{p}$-N,N-didecylaminostyryl)-1-pyridinium]-propylsulfonate. This method is applied to the Ca⁺⁺ mediated fusion of phosphatidyl serine vesicles.     

Detection of programmed cell death using fluorescence energy transfer.

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.