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PubMed1989

Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Fluorescence energy transfer is widely used for determination of intramolecular distances in macromolecules. The time dependence of the rate of energy transfer is a function of the donor/acceptor distance distribution and fluctuations between the various conformations which may occur during the lifetime of the excited state. Previous attempts to recover both distance distributions and segmental diffusion from time-resolved experiments have been unsuccessful due to the extreme correlation between fitting parameters. A method has been developed, based on global analysis of both donor and acceptor fluorescence decay curves, which overcomes this extreme cross-correlation and allows the parameters of the equilibrium distance distributions and intramolecular diffusion constants to be recovered with high statistical significance and accuracy. Simulation studies of typical intramolecular energy transfer experiments reveal that both static and dynamic conformational distribution information can thus be obtained at a single temperature and viscosity.

Biopolymers↗
PubMed2006

Detection of DNA hybridization using induced fluorescence resonance energy transfer.

Induced fluorescence resonance energy transfer (iFRET) is a variation of resonance energy transfer that is particularly well-suited for the detection of DNA hybridization. The underlying mechanism involves monitoring changes in fluorescence that are the result of an energy transfer reaction between a specific pair of donor and acceptor moieties. In iFRET, the donor is a dye that only fluoresces while interacting with double-stranded DNA and the acceptor is dye that is covalently linked to an oligonucleotide probe. Hybridization of the probe to its complement induces excitement of the donor dye and subsequent energy transfer to the acceptor dye. The energy transfer reaction (and concomitant hybridization status) can easily be followed by monitoring the fluorescence output of the acceptor dye. Because the interaction of the donor dye is reversible and dependent on the presence of double-stranded DNA, iFRET is extremely useful and herein demonstrated in the generation of DNA melting curves.

DNA↗
PubMed2001

RNA conformation and folding studied with fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) results from nonradiative coupling of two fluorophores and reports on distances in the range 10-100 A. It is therefore a suitable probe to determine distances in RNA molecules and define their global structure, to follow kinetics of RNA conformational changes during folding in real time, to monitor ion binding, or to analyze conformational equilibria and assess the thermodynamic stability of tertiary structure conformers. Along with the basic principles of steady-state and time-resolved fluorescence resonance energy transfer measurements, approaches to investigate RNA conformational transitions and folding are described and illustrated with selected examples. The versatility of FRET-based techniques has recently been demonstrated by implementations of FRET in high-throughput screening of potential drugs as well as studies of energy transfer that monitor RNA conformational changes on the single-molecule level.

Diagnostic Imaging↗
PubMed2006

Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbium-fluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs.

Fluorescence-based kinase assays using peptide substrates are an established format for high-throughput screening and profiling of kinases. Among fluorescence-based formats, time-resolved fluorescence resonance energy transfer (TR-FRET) using a lanthanide donor species has advantages over other fluorescent formats in being resistant to many types of optical interference such as autofluorescent compounds, scattered light from precipitated compounds, or colored compounds that absorb excitation or emission radiation ("color quenchers"). By taking advantage of the fact that acceptors such as fluorescein or green fluorescent protein (GFP) can be paired with a terbium donor in a TR-FRET assay, we have developed TR-FRET kinase assays that use physiologically relevant native protein substrates, either labeled with fluorescein or expressed as GFP fusions. Phosphorylation of the labeled protein substrate results in an increase in TR-FRET when incubated with a terbium-labeled antibody that specifically recognizes the phosphorylated product. Thus, a strategy of using terbium-based TR-FRET can be applied to develop kinase assays, and the unique properties of terbium lead to a high degree of flexibility with regard to specific assay design.

Animals↗
PubMed1992

Orientation factor in steady-state and time-resolved resonance energy transfer measurements.

Resonance energy transfer measurements provide a way to estimate distances between chromophores attached to different sites of macromolecules. There are two unknowns involved in resonance energy transfer measurements, the distance between two chromophores and their relative orientation. When static orientational disorder exists, the orientation factor, kappa 2, can vary from 0 to 4, leading to considerable uncertainty in estimation of distances. Fluorescence polarization anisotropy measurements can reduce the degree of uncertainty [Dale & Eisinger (1974) Biopolymers 13, 1573]. There may still be substantial error bounds for the average distance measurements. Time-resolved fluorescence measurements provide an "apparent" average distance and distance distribution containing contributions by both distance and orientation. The contribution of orientation to observed "apparent" average distance and distance distribution widths has been estimated for both simulated and real data. With a single unique distance as input in the simulation and with random but static orientation of donor and acceptor, the recovered average distance is very close to that of the input when the input distance is close to or larger than the Förster distance. The recovered width of apparent distance distribution can be substantial and it changes as a function of Förster distance to average distance ratio and as a function of Förster distance. Similar conclusions apply to the case where there is a real distance distribution. Motional averaging of the orientation was simulated by the Monte Carlo method to estimate the contribution of orientation when chromophores have certain degrees of mobility.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed2002

Molecular topography imaging by intermembrane fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) between lipid-linked donor and acceptor molecules in two apposing lipid bilayer membranes is used to resolve topographical features at an intermembrane junction. Efficient energy transfer occurs when the membranes are apposed closely, which creates an image, or footprint, that maps the contact zone and reveals nanometer-scale topographical structures. We experimentally characterize intermembrane FRET by using a supported membrane junction consisting of a glass-supported lipid membrane, onto which a second membrane is deposited by rupture of a giant vesicle. A series of membrane junctions containing different glycolipids (phosphatidylinositol and ganglioside G(M1)), protein (cholera toxin), and lipid-linked polyethylene glycol are studied. The carbohydrate and protein components influence the intermembrane separation. Differential FRET efficiency is clearly distinguishable for each case. Quantitative analysis of the FRET efficiency yields measurements of intermembrane-separation distances that agree precisely with structural data on G(M1) and cholera toxin. The lateral arrangement of molecular species on the membrane surface thus can be discerned by their influence on membrane spacing without the need for direct labeling of the molecule of interest. In the case of polyethylene glycol lipid-containing membrane junctions, imaging by intermembrane FRET reveals spontaneously forming patterns that are not visible in conventional fluorescence images.

Cholera Toxin↗
PubMed1990

Fructose-1,6-bisphosphate aldolase preferentially associates to glyceraldehyde-3-phosphate dehydrogenase in a mixture of cytosolic proteins as revealed by fluorescence energy transfer measurements.

Fluorescence energy transfer measurements were implemented for demonstrating the specific character of the interaction between aldolase and glyceraldehyde-3-phosphate dehydrogenase. The enzymes, labeled with monobromobimane (donor) and fluorescein isothiocyanate (acceptor), respectively, were mixed into a cytosol preparation and energy transfer between the two fluorophores was observed to develop. This observation reflecting a contact between the two enzymes, suggests that despite the presence of a multitude of potential macromolecular partners glyceraldehyde-3-phosphate dehydrogenase and aldolase are capable of recognizing each other in the cytoplasm. The idea that in vivo associations of metabolically sequential enzymes may be of physiological benefits is consistent with this result.

Animals↗
PubMed2004

Kinetics of the structural transition of muscle thin filaments observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer showed that troponin-I changes the position on an actin filament corresponding to three states (relaxed, closed, and open) of the thin filament (Hai et al. (2002) J. Biochem. 131, 407-418). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to position 1, 133, or 181 of troponin-I and Cys-374 of actin on reconstituted thin filaments was measured to follow the transition between three states of the thin filament. When the free Ca(2+) concentration was increased, the transition from relaxed to closed states occurred with a rate constant of approximately 500 s(-1). For the reverse transition, the rate constant was approximately 60 s(-1). When myosin subfragment-1 was dissociated from thin filaments in the presence of Ca(2+) by rapid mixing with ATP, the transition from open to closed states occurred with a single rate constant of approximately 300 s(-1). Light-scattering measurements showed that the ATP-induced myosin subfragment-1 dissociation occurred with a rate constant of approximately 900 s(-1). In the absence of Ca(2+), the transition from open to relaxed states occurred with two rate constants of approximately 400 and approximately 80 s(-1). These transition rates are fast enough to allow the spatial rearrangement of thin filaments to be involved in the regulation mechanism of muscle contraction.

Actin Cytoskeleton↗
PubMed1981

Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.

Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We followed the subunit exchange among filaments both by mixing the donor-acceptor copolymer with unlabeled F actin, and by mixing donor-labeled F actin with acceptor-labeled F actin. The extent of subunit exchange is dependent on ionic conditions. In addition, different kinetics are observed in the two approaches in the presence of excess magnesium ions. The effects of cytochalasin B as a model actin-binding factor were also investigated. One micromolar cytochalasin B reduces the rates of subunit exchange, monomer incorporation and filament depolymerization. At 10 microM cytochalasin B, we detected destabilization of filaments using a morphological assay. The results are discussed in relation to existing models of actin subunit exchange, and to proposed mechanisms of actin-cytochalasin interactions.

Actins↗
PubMed2003

Cytochrome c location in phosphatidylcholine/cardiolipin model membranes: resonance energy transfer study.

Resonance energy transfer between lipid-bound fluorescent probe 3-methoxybenzanthrone as a donor and heme group of cytochrome c as an acceptor has been examined to ascertain the protein disposition relative to the surface of model membranes composed of phosphatidylcholine and cardiolipin (10, 50 and 80 mol%). The model of energy transfer in membrane systems has been extended to the case of donors distributed between the two-bilayer leaflets and acceptors located at the outer monolayer taking into account the donor and acceptor orientational behavior. Assuming specific protein orientation relative to the membrane surface and varying lateral distance of the donor-acceptor closest approach in the range from 0 to 3.5 nm the limits for possible heme distances from the bilayer midplane have been found to be 0.8-3 nm (10 mol% CL), 0-2.6 nm (50 mol% CL), and 1.4-3.3 nm (80 mol% CL).

Animals↗
PubMed1994

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

Adenine Nucleotides↗
PubMed1995

Proximity relationships between engineered cysteine residues in chicken skeletal myosin regulatory light chain. A resonance energy transfer study.

Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys2 and Cys155 and Cys73 and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys2 and Cys155 was measured to be 35 and 30 A in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys73 and Cys155 measured in a similar manner was 31 and 30 A in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.

Animals↗
PubMed1996

Determination of DNA helical handedness by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to determine the helical handedness, twist and rise of different DNA conformations. The approach is based on the construction of a set of molecules consisting of two fused helical segments, one of which is in a known reference helical form. The duplexes are covalently labeled at one end with a donor and at the other with an acceptor. By systematically shifting the position of the junction while maintaining constant the total length in base-pairs, the variation in the efficiency of energy transfer can be shown to depend primarily and sensitively on the differences in helical twist and rise of the two constituent segments. If the latter have the same helical sense, one predicts a FRET signal that is a monotonic function of the junctional position. In contrast, a periodic function arises when two segments are of opposite handedness. The formalism includes explicit consideration of dye orientation (the dipole-dipole orientation factor kappa) and an implementation valid for single helix molecules, and introduces new functions of measured fluorescence signals for establishing the FRET efficiency. The method has been applied to a family of oligonucleotides forming hairpin duplexes containing an antiparallel-stranded (aps) d(m5C.G)m segment labeled at the 5' end with fluorescein (donor) and a second parallel- stranded d(A.T)N-m segment (psAt-DNA) labeled at the hairpin loop with the sulfoindocyanine dye Cy3. The segment lengths were in the range 4 to 12, but the total length N was maintained constant at 16. The d(m5C.G) sequence was chosen due to its capacity for adopting a B or a Z conformation at low and high concentrations of salt, respectively. The parallel-stranded d(A.T) sequence served as the second segment in order to determine the helical rise and twist of psAT-DNA, presumed to be right-handed from molecular modeling and a prior study of topologically constrained DNA. A Z-DNA/ps-DNA junction was created between the two segments by inducing a B-Z transition in d(m5C.G)m with MgCl2. The range of required salt concentration was established by circular dichroism measurements. FRET efficiency values of 0.38 to 0.41 were obtained for the oligonucleotides with the d(m5C.G) segment in the B conformation. In contrast, upon induction of the B-Z transition the FRET efficiency was a decreasing function of the d(m5C.G) content (0.38 to 0.28 for m = 6 to 12). Helical parameters were estimated from functional fits of the data, and were consistent with the known properties of B- and Z-DNAs and with the conclusion that psAT-DNA has a helical rise and twist close to that of B-DNA. The approach outlined here is not restricted to DNA but can be applied to other helical structures, e.g. RNA, proteins, and protein-nucleic acid complexes.

DNA↗
PubMed2000

End-to-end distance distribution in bradykinin observed by Förster resonance energy transfer.

Förster resonance energy transfer (FRET) was used to study the conformational dynamics of bradykinin related peptides. The fluorescent probe aminobenzoic acid (Abz) bound to the amino terminal of bradykinin maintained its fluorescence characteristics, like high quantum yield and excited state decay dominated by a lifetime of 8.3 ns. The binding of the acceptor group N-[2, 4-dinitrophenyl]-ethylenediamine (EDDnp) to the carboxy terminal of Abz labeled bradykinin resulted in a drastic decrease of the fluorescence intensity and in a fastening of the excited state decay. The change of the decay kinetics to an heterogeneous process, precludes the use of energy transfer models based on a single fixed distance between donor and acceptor. The computational package CONTIN was employed to the analysis of time-resolved fluorescence data, allowing the recovery of a distance distribution between donor and acceptor corresponding to the end-to-end distance of the labeled peptide. The distance distribution reflects the occurrence of distinct conformations for the peptide, that coexist in equilibrium during the fluorescence lifetime. We observed three distance populations for bradykinin in water, that merged to two populations when the solvent was trifluoroethanol (TFE). The results were consistent with those obtained from circular dichroism spectroscopy, that showed structural flexibility in water and the presence of more defined secondary structure in TFE. We also studied several peptides related to bradykinin, and the results emphasized the formation of turns involving the proline residues and the decrease of conformational flexibility induced by using TFE as the solvent.

Amino Acid Sequence↗
PubMed2002

Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer.

Luminescence resonance energy transfer with a lanthanide like Tb(3+) as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually requires the use of a bulky chelator with a flexible linker attached to a Cys residue to bind Tb(3+) and, for intramolecular studies, an acceptor fluorophor attached to another Cys residue in the same protein. Here, an engineered EF- hand motif is incorporated into the central cytoplasmic loop of the lactose permease of Escherichia coli generating a high-affinity site for Tb(3+) (K(Tb)(3+) approximately 4.5 microM) or Gd(3+) (K(Gd)(3+) approximately 2.3 microM). By exciting a Trp residue in the coordination sequence, Tb(3+) bound to the EF-hand motif is sensitized specifically, and the efficiency of energy transfer to strategically placed Cys residues labeled with fluorophors is measured. In this study, we use the technique to measure distance from the EF-hand in the central cytoplasmic loop of lactose permease to positions 179 or 169 at the center or periplasmic end of helix VI, respectively. The average calculated distances of approximately 23 A (position 179) and approximately 33 A (position 169) observed with three different fluorophors as acceptors agree well with the geometry of a slightly tilted alpha-helix. The approach should be of general use for studying static and dynamic aspects of polytopic membrane protein structure and function.

Binding Sites↗
PubMed1995

On the revised concept of linear energy transfer.

The quantities linear energy transfer or restricted linear energy transfer are utilized in calculations that link absorbed dose to the fluence distribution of a radiation field. The computations provide approximations to absorbed dose in terms of the intermediate quantity cema or reduced cema. With the definition of the restricted linear energy transfer, L delta, given in ICRU Report 33, the approximation remains imperfect. This study deals with the resulting need for a modified definition of L delta, as proposed in a draft report of ICRU. Essential differences between the old and the new definitions are demonstrated. The changed definition permits a rigorous formulation of the dependence between fluence and absorbed dose.

Dose-Response Relationship, Radiation↗
PubMed1983

Cytosol-membrane interface of human erythrocytes. A resonance energy transfer study.

The resonance energy transfer from donors embedded in the membrane of erythrocytes to the cytosol hemoglobin has been measured by comparing the donors' fluorescence decay in ghosts and in intact cells. A series of n - (9-anthroyloxy) stearic acids (n-AS) (n = 2, 6, 9, 12) and similar probes were used as donors, and their locations within the outer leaflet of the phospholipid bilayer were determined from their average efficiency of energy transfer, . The energy transfer data for several membrane probes were analyzed according to a simple semiempirical model, in which the heme acceptors are assumed to form a semiinfinite continuum beyond a plane, whose normal distance (d) from particular donors may be determined if the heme density in the cytosol boundary layer is known. The hemoglobin concentration in the erythrocytes was varied by suspending the cells in buffers of different ionic strengths. This made it possible to study the ionic strength dependence of the heme concentration averaged over the cell (h(c)), as well as that in the boundary layer (h(b)). Both level off above approximately 600 mosM, as does the ratio h(b)/h(c). By using the maximum heme concentration that can be obtained in osmotically shrunken cells as a limiting value, h(b) is estimated to be 17 mM or less, under physiological conditions; and from the measured for various probes, the distance d was found to range from 40 A for 2-AS to 31 A for 12-AS and 26 A for 9-vinyl anthracene (9-VA). It is concluded that the hydrophobic probe 9-VA is located near the center of the phospholipid bilayer and that the cytosol hemoglobin is in contact with the inner membrane surface, or nearly so. This conclusion is valid for oxy- and deoxy-hemoglobin, and is shown to be independent of several systematic errors that might arise from the simple assumptions of the model used. The steady-state fluorescence anisotropy of the probes was found to decrease as they approach the bilayer's central plane. The methodology developed here may be used to extend studies of cytosol membrane interactions in ghost systems to intact cells, and is useful in the investigation of the morphology of normal and pathological intact erythrocytes.

Cytosol↗
PubMed1989

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer.

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for Förster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.

Bacteriorhodopsins↗