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PubMed1989

Determination of the fractal dimension of membrane protein aggregates using fluorescence energy transfer.

It is demonstrated that fluorescence resonance energy transfer may be used to determine the fractal dimension of aggregates of membrane-bound proteins. Theoretical and experimental results are presented for two different experimental designs: energy transfer between proteins and energy transfer from lipids to proteins. For energy transfer between proteins the lattice spacing must be known independently for a fractal dimension to be uniquely determined, and this represents a disadvantage to this experimental design. Results are presented for the calcium ATPase and a fractal dimension of 1.9 is estimated for ATPase aggregates by assuming a lattice spacing of 50 A. Energy transfer from lipids to protein provides a means of estimating the length of the "coast-line" of the aggregate. In this case the fractal dimension is uniquely determined from a log-log plot. An analysis of data for bacteriohodopsin reconstituted in phospholipid vesicles gives a fractal dimension of 1.6. The structural basis of the value for the fractal dimension is discussed for these two systems. These techniques provide a means of assessing the nature of protein-protein interactions in membranous systems.

Calcium-Transporting ATPases↗
PubMed1998

Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.

A Trp-free alphaA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between alphaA- and alphaA-crystallins as well as between alphaA- and alphaB-crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type alphaA- or alphaB-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37 degrees C than at 25 degrees C. The energy transfer efficiency was greater between homogeneous subunits (alphaA-alphaA) than between heterogeneous subunits (alphaA-alphaB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alphaA and alphaB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alphaA-alphaA and alphaA-alphaB indicated that alphaA- and alphaB-crystallins were not necessarily structurally equivalent.

Anilino Naphthalenesulfonates↗
PubMed2002

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.

Biophysical Phenomena↗
PubMed1988

Time-resolved detection of energy transfer: theory and application to immunoassays.

Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to separate the component of acceptor emission due to energy transfer from the component due to absorption of the excitation light. Theoretical equations describing the acceptor fluorescence and integrated acceptor fluorescence show that increasing the integration delay relative to the excitation pulse should greatly enhance detection of the energy-transfer component. The time-resolved detection of energy transfer was tested in a competitive immunoassay format in which antibodies to human immunoglobulin G (IgG) F(ab')2 fragments were covalently labeled with pyrenebutyrate (tau = 100 ns) and IgG Fab' fragments were covalently labeled with B-phycoerythrin (tau = 2.5 ns). Solutions containing these two conjugates exhibited energy transfer from the pyrenebutyrate to the B-phycoerythrin upon excitation with a nitrogen laser. Acceptor emission was measured with 0- and 20-ns integration delays and the ratios of the energy-transfer component to the laser-excited component were found to increase by 9- to 15-fold when the 20-ns delay was used in three series of immunoassays. Good agreement between the experimental data and theory was obtained following convolution of the theoretical fluorescence responses with the instrumental response of the fluorometer.

Antigens↗
PubMed1992

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Excitation energy transfer from phycobilisomes to photosystems: a phenomenon associated with the temporal separation of photosynthesis and nitrogen fixation in a cyanobacterium, Plectonema boryanum(1).

Plectonema boryanum shows temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. Low temperature fluorescence studies have shown that in vivo the nitrogen fixing and photosynthesizing cells are adapted to 'state 2' and 'state 1', respectively. During nitrogen fixation phycobilisomes seem to transfer excitation energy to photosystem I whereas during oxygenic photosynthesis the energy is transferred to photosystem II. The state 2 adapted N-phase cells failed to undergo transition to state 1 while P-phase cells exhibited state 1 to state 2 transition. The nitrogen fixing cells showed a decreased level of psbC transcript, lack of CP47 in thylakoid membrane, and presence of the F685 peak but absence of the F695 peak in 77 K fluorescence spectra. These results suggest that the metabolic and molecular changes associated with nitrogen fixation may favor direct energy transfer from the phycobilisomes to photosystem I. This should help the organism to achieve low photosystem II and high photosystem I activity to set temporal separation of nitrogen fixation and photosynthesis for photoautotrophic growth under diazotrophic conditions.

Journal Article↗
PubMed2005

Temperature dependent energy transfer in Ar-O3 collisions.

The energy transfer between argon atoms and ozone complexes O3*, excited in the region of the dissociation threshold, is calculated for fixed temperatures (100 K< or =T < or =2500 K) using classical trajectories. The internal energy of ozone is resolved in terms of vibrational and rotational energies. For all temperatures, energy flows from O3* to Ar. The vibrational energy transfer, relative to k(B)T, is very small below 500 K, but gradually increases towards high temperatures. The relative rotational energy transfer, on the other hand, monotonously decreases with T; around 1100 K it falls below the relative vibrational energy transfer. Thermally averaged cross sections for vibrational and rotational energy transfers are also calculated. The implications for the stabilization of ozone complexes in the energy transfer model are discussed.

Journal Article↗
PubMed1983

Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAphe.

The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules. Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe. The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17). The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions. Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A. Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees.

Anticodon↗
PubMed2006

Energy transfer in the major intrinsic light-harvesting complex from Amphidinium carterae.

Carbonyl carotenoids are important constituents of the antenna complexes of marine organisms. These carotenoids possess an excited state with a charge-transfer character (intramolecular charge transfer state, ICT), but many details of the carotenoid to chlorophyll energy transfer mechanisms are as yet poorly understood. Here, we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the intrinsic light-harvesting complex (LHC) of dinoflagellates, which contains the carbonyl carotenoid peridinin. Carotenoid to chlorophyll energy transfer efficiency is about 90% in the 530-550 nm region, where the peridinin S2 state transfers energy with an efficiency of 25-50%. The rest proceeds via the S1/ICT channel, and the major S1/ICT-mediated energy transfer pathway utilizes the relaxed S1/ICT state and occurs with a time constant of 2.6 ps. Below 525 nm, the overall energy transfer efficiency drops because of light absorption by another carotenoid, diadinoxanthin, that contributes only marginally to energy transfer. Instead, its role is likely to be photoprotection. In addition to the peridinin-Chl-a energy transfer, it was shown that energy transfer also occurs between the two chlorophyll species in LHC, Chl-c2, and Chl-a. The time constant characterizing the Chl-c2 to Chl-a energy transfer is 1.4 ps. The results demonstrate that the properties of the S1/ICT state specific for carbonyl carotenoids is the key to ensure the effective harvesting of photons in the 500-600 nm region, which is of vital importance to underwater organisms.

Carotenoids↗
PubMed2005

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.

Cell Line, Tumor↗
PubMed2005

Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer.

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

Bacterial Proteins↗
PubMed2006

Spectrally resolved frequency domain analysis of multi-fluorophore systems undergoing energy transfer.

Complex systems of fluorophores undergoing energy transfer can exhibit a variety of anomalous lifetime behavior when probed with frequency domain methods. When presented in traditional apparent lifetime format the data from such systems can exhibit "nodal" behavior in which the computed lifetime approaches +/-infinity. The location of the nodes is system and frequency dependent. In addition, simpler systems, not undergoing energy transfer, show ill behavior in the region of zero lifetime (tau(m)) and long lifetime (tau(pi)) due to noise in typical measurements. Here, we systematically investigate systems of multiple fluorophores with and without energy transfer to provide insight into frequency domain investigations of complex systems of fluorophores. The results of simulations are compared to data collected from a multi-fluorophore system designed to exhibit fluorescence resonance energy transfer (FRET) using imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM). The results are applicable to both cuvette and imaging arrangements.

Algorithms↗
PubMed2001

Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa extracellular matrix protein, with functional sites that are regulated by unfolding events. Fn was labeled with multiple donor and acceptor fluorophores such that intramolecular FRET could be used to distinguish a range of Fn conformations. The sensitivity of FRET to unfolding was tested by progressively denaturing labeled Fn using guanidium chloride. To investigate Fn conformation changes during cell binding and matrix assembly, we added labeled Fn to the culture medium of NIH 3T3 fibroblasts. Coexisting conformations of Fn were visualized using fluorescence microscopy, and spectra from specific features were measured with an attached spectrometer. Using FRET as an indicator of Fn conformation, Fn diffusely bound to cells was in a compact state, whereas Fn in matrix fibrils was highly extended. Matrix fibrils exhibited a range of FRET that suggested some degree of unfolding of Fn's globular modules. Fn in cell-associated clusters that preceded fibril formation appeared more extended than diffuse cell-bound Fn but less extended than fibrillar Fn, suggesting that Fn undergoes extension after cell binding and before polymerization. FRET thus provides an approach to gain insight into the integrin-mediated pathway of Fn fibrillogenesis.

3T3 Cells↗
PubMed2001

Structural study of DNA duplexes containing the (6-4) photoproduct by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.

Cisplatin↗
PubMed2006

Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology.

We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.

Biotechnology↗
PubMed1989

Visualization of epidermal growth factor (EGF) receptor aggregation in plasma membranes by fluorescence resonance energy transfer. Correlation of receptor activation with aggregation.

Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in these energy transfer measurements, while EGF molecules labeled with eosin isothiocyanate at the amino terminus served as the energy acceptors. Both of these derivatives were shown to be active in binding to membrane receptors and in the activation of the endogenous receptor/tyrosine kinase activity. We found that membranes in the absence of added metal ion activators showed relatively little energy transfer (approximately 10% donor quenching) between the labeled growth factors. However, divalent metal ion activators of the EGF receptor/tyrosine kinase caused a significant increase in the extent of energy transfer between the labeled EGF molecules. Specifically, in the presence of 20 mM MgCl2, the extent of quenching of the donor fluorescence increased to 25% (from 10% in the absence of metal), while in the presence of 4 mM MnCl2, the extent of energy transfer was increased still further to 40-50%. The addition of an excess of EDTA resulted in the reversal of the observed energy transfer to basal levels. The increased energy transfer in the presence of these divalent cations correlated well with the ability of these metals to stimulate the EGF receptor/tyrosine kinase activity. However, the extent of receptor-receptor interactions measured by energy transfer was independent of receptor autophosphorylation. Overall, these results suggest that conditions under which the EGF receptor is primed to be active as a tyrosine kinase, within a lipid milieu, result in an increased aggregation of the receptor.

Binding, Competitive↗
PubMed1977

Energy transfer and the distribution of excitation energy in the photosynthetic apparatus of spinach chloroplasts.

Equations are derived from our model of the photochemical apparatus of photosynthesis to show that the yield of energy transfer from Photosystem II to Photosystem I, phi T(II leads to I), can be obtained from measurements on an individual sample of chloroplasts frozen to -196 degrees C by comparing the sum of two specifically defined fluorescence excitation spectra with the absorption spectrum of the sample. Then, given that value of phiT(II leads to I), the fraction of the quanta absorbed by the photochemical apparatus which is distributed initially to Photosystem I, alpha, can be determined as a function of the wavelength of excitation from the same fluorescence excitation spectra. The results obtained in this study of individual samples of chloroplasts frozen to -196 degrees C in the absence of divalent cations, namely, that phi T(II leads to I)varies from a minimum value of 0.10 when the Photosystem II reaction centers are all open to a maximum value of 0.25 when the centers are all closed and that alpha has a value of about 0.30 which is almost independent of wavelength for wavelength shorter than 675 nm (alpha increases rapidly toward unity at wavelength longer than 675 nm), agrees quite well with results obtained previously from comparative measurements of chloroplasts frozen to -196 degrees C in the presence and absence of divalent cations.

Chloroplasts↗
PubMed2001

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.

Animals↗