Analysis of resonance energy transfer in model membranes: role of orientational effects

The model of resonance energy transfer (RET) in membrane systems containing donors randomly distributed over two parallel planes separated by fixed distance and acceptors confined to a single plane is presented. Factors determining energy transfer rate are considered with special attention being given to the contribution from orientational heterogeneity of the donor emission and acceptor absorption transition dipoles. Analysis of simulated data suggests that RET in membranes, as compared to intramolecular energy transfer, is substantially less sensitive to the degree of reorientational freedom of chromophores due to averaging over multiple donor-acceptor pairs. The uncertainties in the distance estimation resulting from the unknown mutual orientation of the donor and acceptor are analyzed.     

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.     

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.     

Engineering resonance energy transfer for advanced immunoassays: the case of celiac disease

Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor–acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Structural information on nanomolecular systems revealed by FRET

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D-A distance distribution function rhoDA(r). We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers.     

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer

BACKGROUND: DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. METHODS: We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). RESULTS: The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. CONCLUSIONS: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.     

Anisotropic excitation transfer to acceptors randomly distributed on surfaces.

We presented exact expressions for the ensemble averaged decay of the excitation of a donor molecule due to the energy transfer via anisotropic dipolar interactions to acceptors distributed randomly on a surface. The disorder extended both over the positions of the acceptors and over the orientations of their transition dipoles with respect to that of the donor molecule. Several cases were considered explicitly (a) random orientations of the acceptors in space, with the donor being (a1) perpendicular to the plane, (a2) in the plane, (a3) randomly oriented in space; (b) random orientations of both donor and acceptors in the plane; (c) parallel orientations of donor and acceptors (no orientational disorder). For all these cases we evaluated the analytic, Förster-like expressions, valid for long times and low acceptor densities, and obtained their domains of validity by comparison with the exact, numerically calculated decay laws.     

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.     

On the possibility of long-wavelength long-lifetime high-quantum-yield luminophores

We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.     

The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 ± 0.08 and 6.2 ± 0.03.     

Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion

Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.     

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 transfer activity in synchronous populations of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis employing steady-state synchronously dividing populations of Rhodobacter sphaeroides reveal that the translocation of pre-existing phospholipid into the growing membrane is concurrent with cell division (Cain, B.D., Deal, C.D., Fraley, R.T. and Kaplan, S. (1981) J. Bacteriol. 145, 1154–1166), yet the mechanism of phospholipid movement is unknown. However, the discovery of phospholipid transfer protein activity in R. sphaeroides (Cohen, L.K., Lueking, D.R. and Kaplan, S. (1979) J. Biol. Chem. 254, 721–728) provides one possible mechanism for phospholipid movement. Therefore the level of phospholipid transfer activity in cell lysates of synchronized cultures was measured and was shown to increase stepwise coinciding precisely with the increase in cell number of the culture. Although the amount of transfer activity per cell remained constant throughout the cell cycle, the specific activity of the phospholipid transfer activity showed a cyclical oscillation with its highest value coincident with the completion of cell division. Purified intracytoplasmic membrane can be used as phospholipid acceptor in the developed phospholipid transfer assay by employing either cytoplasmic membrane or liposomes as the phospholipid donor. Intracytoplasmic membrane isolated from the cells prior to division (high protein to phospholipid ratio) served as a better phospholipid acceptor in the phospholipid transfer system when compared with membranes derived from the cells following cell division (low protein to phospholipid ratio).     

Biosynthesis of the polysialic acid capsule in Escherichia coli K1. The endogenous acceptor of polysialic acid is a membrane protein of 20 kDa

The nature of endogenous acceptor molecules implicated in the membrane-directed synthesis of the polysialic acid (polySia) capsule in Escherichia coli K1 serotypes is not known. The capsule contains at least 200 sialic acid (Sia) residues that are elongated by the addition of new Sia residues to the nonreducing termini of growing nascent chains (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342). Presumably, chain growth starts when activated Sia residues are transferred to acceptors that are not already sialylated. In the present study, we used an acapsular mutant defective in synthesis of CMP-NeuAc to label acceptors with [14C]NeuAc and an anti-polySia-specific antibody (H.46) to identify the molecules to which the polySia was attached. [14C]Sia-labeled acceptors were solubilized with 2% Triton X-100, immunoprecipitated with H.46, and partially depolymerized with poly-alpha-2,8-endo-N-acetylneuraminidase. Approximately 5% of the [14C]Sia incorporated remained attached to endogenous acceptors. Double-labeling experiments were used to show that the non-Sia moiety of the acceptor was labeled in vivo with [14C]leucine and elongated in vitro with CMP-[3H]NeuAc. Concomitant with desialylation of the [3H]polySia-[14C]Leu acceptor was the appearance of a new [14C]Leu-labeled protein at 20 kDa. After strong acid hydrolysis, the 20-kDa labeled protein was shown to contain [14C]Leu. The acceptor molecules were not labeled metabolically with D-[3H]GlcN, 35SO4, or 32PO4, indicating that they do not appear to contain lipopolysaccharide, peptidoglycan, phosphatidic acid, or phospholipid. Based on these results, we conclude that the endogenous acceptor molecule is a membrane protein of about 20 kDa. The nature of attachment of polySia to acceptor is unknown. There are only 400-500 acceptor molecules/cell, which is about 100-fold fewer than the 50,000 polySia chains/cell. This suggests that each acceptor molecule may participate in the shuttling of about 100 polySia chains/cell. We hypothesize that the acceptor protein may function to translocate polySia chains from their site of synthesis on the cytoplasmic surface of the inner membrane to the periplasm.     

Texture analysis of fluorescence lifetime images of nuclear DNA with effect of fluorescence resonance energy transfer

BACKGROUND: Fluorescence lifetime imaging microscopy (FLIM) is becoming an important tool in cellular imaging. In FLIM, the image contrast is concentration insensitive, whereas it is sensitive to the local environment and interactions of fluorophores such as fluorescence resonance energy transfer (RET). METHODS: Fluorescence microscopy, lifetime imaging, and texture analysis were used to study the spatial distribution of fluorophores bound to nuclear DNA. 3T3-Swiss albino mice fibroblast nuclei were labeled with Hoechst 33258 (Ho), an AT-specific dye, and 7-aminoactinomycin D (7-AAD), a GC-specific dye. Ho is a RET donor to the 7-AAD acceptor. RESULTS: Texture analysis of 50 alcohol-fixed nuclei quantitatively showed changes of spatial distribution of apparent donor lifetimes. RET increased the spatial heterogeneity in the phase and modulation lifetime images. In most of the doubly stained cells (about 80%), the phase and modulation lifetime distributions were spatially homogeneous. In about 20% of the cells, we noticed that lower phase and modulation lifetimes caused by RET were correlated with regions of high Ho intensity in the nuclei. CONCLUSIONS: The spatial lifetime heterogeneity of Ho in presence of 7-AAD seems to be caused by RET between closely spaced strands in the three dimensionally condensed regions of DNA.     

Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R(0) near 50 A and a donor to acceptor distance near 75 A. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steady-state and time-resolved measurements. The apparent R(0) in the presence of silver island films increases to about 110 A. These values of the transfer efficiency and R(0) represent weighted averages for donor-acceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces.     

Effects of metallic silver particles on resonance energy transfer in labeled bovine serum albumin

Resonance energy transfer (RET) is widely used to detect proximity between biomolecules. In transparent solution the maximum donor-to-acceptor distance for RET is about 70 A. We measured the effects of metallic silver island films on RET from the intrinsic tryptophan of a protein to a bound probe as the acceptor. These preliminary experiments revealed a dramatic increase in the apparent F?rster distance increasing from 28.6 to 63 A. These results suggest the use of silver island films for detecting long range proximity between biomolecules and for biotechnology applications based on RET.     

Lectin binding to complex carbohydrate at the interface: a study by resonance energy transfer.

The binding affinity of the oligosaccharide moiety of a neutral glycosphingolipid, asialoGM1, towards Ricinus communis agglutinin (RCAI) was determined for the first time by fluorescence resonance energy transfer (RET). The asialoGM1 was incorporated into a phospholipid (DMPC) vesicle doped with dansylated DPPE and then titrated with an increasing amount of the galactose specific RCAI. The efficiency of RET was determined by a saturable increase in the quenching of 'donor' fluorescence, i.e. the 'trp' residue of RCAI, due to the energy transfer from the 'acceptor' dansyl group on the surface of the vesicle. The apparent binding constant was found to be in the range of 10(5)-10(6) M-1 at 27 degrees C.