Application of radiationless energy transfer for distance measurements across membranes

For several 2- or 3-dimensional configurations of stationary donors and acceptors on or near a spherical membrane shell the transition probabilities for radiationless energy transfer are calculated, using Förster's approximation obtained for Coulombic dipole-dipole interaction of the transition moments. It turns out that the difference in the refractive indices for membrane (lipid) and bulk phase (water) has only a small influence on the transition probabilities. Furthermore, the curvature of biological cell surfaces can be neglected, but affects the energy transfer across small vesicles. The ratio thickness/radius of small vesicles can be determined by measuring fluorescence quenching of excited donors by acceptors on the other side of the membrane.     

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).     

Fluorescence resonance energy transfers measurements on cell surfaces via fluorescence polarization

A method has been developed for the determination of the efficiency of fluorescence resonance energy transfer efficiency between moieties located on cell surfaces by performing individual cell fluorescence polarization (FP) measurements. The absolute value of energy transfer efficiency (E) is calculated on an individual cell basis. The examination of this methodology was carried out using model experiments on human T lymphocyte cells. The cells were labeled with fluorescein-conjugated Concanavalin A (ConA) as donor, or rhodamine-conjugated ConA as acceptor. The experiments and results clearly indicate that determination of E via FP measurements is possible, efficient, and more convenient than other methods.     

Transport of Fluorescence through Highly Scattering Media: Corrections to the Determination of Quantum Yields

The equations for transport of fluorescence, generated within a highly scattering medium, are solved within the boundary conditions of the Kubelka and Munk treatment. Expressions are derived in closed form for fluorescence fluxes emanating from the front and back surfaces of a highly scattering infinite slab, whether or not fluorescence is absorbed within the sample. An “apparent” quantum yield, calculated from observed intensities of fluorescence and of back-scattered light from the front surface of the slab, can be corrected by these expressions to return the true quantum yield of fluorescence. Allowance for re-emission of reabsorbed fluorescence can be made, but may not be applicable in some experimental arrangements. Calculations performed on the fluorescence of rhodamine 101 suggest that in typical practical situations the correction factor may not be far from unity.     

What causes the depolarization of trypsin and trypsinogen fluorescence: Intramolecular mobility or non-radiative energy transfer?

Analysis of protein data bank information about the coordinates of definite atoms of protein macromolecules provides an opportunity to evaluate the efficiency of non-radiative resonance energy transfer within the model of fixed, strictly oriented oscillators. Such evaluations for trypsin and trypsinogen (and also for trypsin complex with a pancreatic inhibitor) show that the efficiency of energy transfer among each pair of tryptophan residues is negligibly small. It is also shown that a fairly effective energy transfer from tyrosine to tryptophan residues is possible. The conclusions have been made that the Tyr-Trp energy transfer is one of the factors determining the shape of the trypsin polarization spectrum, and that upon fluorescence excitation at the long-wavelength edge of the absorption spectrum, the depolarization of trypsin fluorescence in aqueous solution at ambient temperature - compared to model compounds (tryptophan, N-acetyltryptophan, glycyltryptophan, etc.), under the conditions of infinite viscosity - is due to the Brownian rotational motion of the macromolecules as a whole as well as the intramolecular mobility. The differences in the level and character of intramolecular mobility of trypsin and trypsinogen are discussed.     

Förster-type energy transfer. Simultaneous 'forward' and 'reverse' transfer between unlike fluorophores

The general case of F?rster-type energy transfer is that in which energy is exchanged in both directions between two unlike fluorophores. In such cases, energy is transferred from the conventionally defined donor to the conventionally defined acceptor (forward transfer) and at the same time from the acceptor to the donor (reverse transfer). Expressions are derived to describe the fluorescence intensities and lifetimes of fluorophores undergoing simultaneous forward and reverse transfer; these are compared with corresponding quantities for the case more usually considered, in which only forward transfer is significant. It is shown that the presence of reverse transfer removes the distinction between donor and acceptor, and allows such anomalous effects as 'acceptor quenching'. A confirmatory example is described. It is shown that the equations generally used in distance determination by steady-state fluorescence spectroscopy can also be applied in the presence of reverse transfer, if a correction term is included; however, for lifetime spectroscopy the correction is more complex.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.     

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.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

Effect of membrane microheterogeneity and domain size on fluorescence resonance energy transfer

Studies of multicomponent membranes suggest lateral inhomogeneity in the form of membrane domains, but the size of small (nanoscale) domains in situ cannot be determined with current techniques. In this article, we present a model that enables extraction of membrane domain size from time-resolved fluorescence resonance energy transfer (FRET) data. We expand upon a classic approach to the infinite phase separation limit and formulate a model that accounts for the presence of disklike domains of finite dimensions within a two-dimensional infinite planar bilayer. The model was tested against off-lattice Monte Carlo calculations of a model membrane in the liquid-disordered (l(d)) and liquid-ordered (l(o)) coexistence regime. Simulated domain size was varied from 5 to 50 nm, and two fluorophores, preferentially partitioning into opposite phases, were randomly mixed to obtain the simulated time-resolved FRET data. The Monte Carlo data show clear differences in the efficiency of energy transfer as a function of domain size. The model fit of the data yielded good agreement for the domain size, especially in cases where the domain diameter is <20 nm. Thus, data analysis using the proposed model enables measurement of nanoscale membrane domains using time-resolved FRET.     

Gibbs free energy of transfer of glycine and diglycine from water to alkylurea solutions. Measured vs. calculated values

Ternary systems comprising water (1), glycine (diglycine) (2) and alkylurea (3) have been investigated using vapor pressure osmometry. Equations were obtained in terms of the molalities of the solutes for the activity coefficients of glycine and diglycine in these systems. The alkylureas used were methyl-, ethyl- and N, N'-dimethylurea. Using the activity coefficients the Gibbs free energy of transfer at infinite dilution of component 2 from water to alkylurea solutions was determined. Since the enthalpies of transfer are known, the corresponding entropies could also be obtained. Calculation of the Gibbs free energy of transfer at infinite dilution of component 2 rests on the assumption that it can be divided into two parts: the difference between the Gibbs free energy of cavity formation and that of interaction in the alkylurea solution and water, respectively. The first part was calculated by scaled particle theory using experimental density and surface tension data. The second part was taken to be due mainly to the change in dipole-dipole interactions.     

Differential transform semi-numerical analysis of biofluid-particle suspension flow and heat transfer in non-Darcian porous media

The differential transform method (DTM) is semi-numerical method which is used to study the steady, laminar buoyancy-driven convection heat transfer of a particulate biofluid suspension in a channel containing a porous material. A two-phase continuum model is used. A set of variables is implemented to reduce the ordinary differential equations for momentum and energy conservation (for both phases) to a dimensionless system. DTM solutions are obtained for the dimensionless system under appropriate boundary conditions. We examine the influence of momentum inverse Stokes number (Sk_m), Darcy number (Da), Forchheimer number (Fs), particle loading parameter (p_L), particle-phase wall slip parameter (Ω) and buoyancy parameter (B) on the fluid-phase velocity (U) and particle-phase velocity (U_p). Padé approximants are also employed to achieve satisfaction of boundary conditions. Excellent correlation is obtained between the DTM and numerical quadrature solutions. The results indicate that there is a strong decrease in fluid-phase velocities with increasing Darcian (first-order) drag and the second-order Forchheimer drag, and a weaker reduction in particle-phase velocity field. Fluid and particle-phase velocities are also strongly affected with inverse momentum Stokes number. DTM is shown to be a powerful tool providing engineers with an alternative simulation approach to other traditional methods for multi-phase computational biofluid mechanics. The model finds applications in haemotological separation and biotechnological processing.     

Calculation of resonance energy transfer in crowded biological membranes.

Analytical and numerical models were developed to describe fluorescence resonance energy transfer (RET) in crowded biological membranes. It was assumed that fluorescent donors were linked to membrane proteins and that acceptors were linked to membrane lipids. No restrictions were placed on the location of the donor within the protein or the partitioning of acceptors between the two leaflets of the bilayer; however, acceptors were excluded from the area occupied by proteins. Analytical equations were derived that give the average quantum yield of a donor at low protein concentrations. Monte Carlo simulations were used to generate protein and lipid distributions that were linked numerically with RET equations to determine the average quantum yield and the distribution of donor fluorescence lifetimes at high protein concentrations, up to 50% area fraction. The Monte Carlo results show such crowding always reduces the quantum yield, probably because crowding increases acceptor concentrations near donor-bearing proteins; the magnitude of the reduction increases monotonically with protein concentration. The Monte Carlo results also show that the distribution of fluorescence lifetimes can differ markedly, even for systems possessing the same average lifetime. The dependence of energy transfer on acceptor concentration, protein radius, donor position within the protein, and the fraction of acceptors in each leaflet was also examined. The model and results are directly applicable to the analysis of RET data obtained from biological membranes; their application should result in a more complete and accurate determination of the structures of membrane components.     

Microarray analysis of protein-protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime imaging

The detection of protein-protein binding on microarrays using the fluorescence lifetime as a dynamic analytical parameter was investigated in a model system. The assay is based on F?rster resonance energy transfer (FRET) and carried out with biotinylated Bovine Serum Albumin and streptavidin, labeled with the commonly used microarray dyes Alexa 555 and Alexa 647, respectively. This efficient FRET donor/acceptor pair was employed in a competitive assay format on three different microarray surfaces. The fluorescence was excited by 200ps laser pulses from a mode-locked and cavity-dumped argon-ion laser, adapted to an intensified CCD camera as detection unit allowing time resolution with subnanosecond precision. Lifetime maps were recorded according to the Rapid Lifetime Determination (RLD) scheme. Interaction between the proteins could clearly be detected on all formats and resulted in almost complete quenching on CEL Epoxy surfaces upon addition of excess streptavidin labeled the FRET acceptor dye. In this case, the fluorescence lifetimes dropped by 90%, whereas on ARChip Epoxy and ARChip Gel the reduction was 54% and 47%, respectively. Good linearity of the quenching curve was obtained in all cases. The method is applicable to all types of protein interaction analysis on microarrays, particularly in cases where evaluation of fluorescence intensity is prone to erroneous results and a more robust parameter is required.     

Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

Interaction between beta-amyloid and lens alphaB-crystallin

In Alzheimer's disease, beta-amyloid peptides (betaA(1-40) and betaA(1-42)) are deposited on the brain cell surfaces as neurotoxic plaques. Some reports indicate that small heat shock proteins, Hsp27 and alphaB-crystallin, colocalize in the plaques, but their functions are not known. Interaction between betaA and alphaB-crystallin must be determined in order to understand the role of alphaB-crystallin in betaA fibril formation. We used a pyrene (Pyr)-labeled betaA(1-40) in a fluorescence energy transfer experiment. Upon incubation together at 37 degrees C, energy transfer between Trp of alphaB-crystallin and Pyr of Pyr-labeled betaA was observed, indicating that betaA participated in subunit exchange of alphaB-crystallin, which promoted fibril formation.     

Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy

Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.     

Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin

The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three‐dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non‐radiation energy transfer. The quenching constants K_SV, binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non‐radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX–pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study. Copyright © 2013 John Wiley & Sons, Ltd.