Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb3+ fluorescent chelate with N,N,N',N'-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb3+) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb3+ labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb3+ labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ng ml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement. (C) 2001 Elsevier Science B.V. All rights reserved.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Distance dependence of fluorescence resonance energy transfer

Deviations from the usual R (-6) dependence of the rate of fluorescence resonance energy transfer (FRET) on the distance between the donor and the acceptor have been a common scenario in the recent times. In this paper, we present a critical analysis of the distance dependence of FRET, and try to illustrate the non R (-6) type behaviour of the rate for the case of transfer from a localized electronic excitation on the donor, a dye molecule to three different energy acceptors with delocalized electronic excitations namely, graphene,two-dimensional semiconducting sheet and the case of such a semiconducting sheet rolled to obtain a nanotube. We use simple analytic models to understand the distance dependence in each case.

Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer

The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM(1)) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

Fluorescence enhancement and energy transfer in apertureless scanning near-field optical microscopy

We investigate the mechanisms for fluorescence enhancement and energy transfer near a gold tip in apertureless scanning near-field optical microscopy. Using a simple quasi-static model, we show that the observed enhancement of fluorescence results from competition between enhancement and quenching, and is dependent on a range of experimental parameters. We find good qualitative agreement with the results of measurements of the effect of both sharp and blunt tips on quantum dot fluorescence, and provide a demonstration of tip-enhanced fluorescence imaging with 60 nm resolution.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.

Energy transfer to a proton-transfer fluorescence probe: Tryptophan to a flavonol in human serum albumin

A protein fluorescence probe system, coupling excited-state intermolecular Förster energy transfer and intramolecular proton transfer (PT), is presented. As an energy donor for this system, we used tryptophan, which transfers its excitation energy to 3-hydroxyflavone (3-HF) as a flavonol prototype, an acceptor exhibiting excited-state intramolecular PT. We demonstrate such a coupling in human serum albumin–3-HF complexes, excited via the single intrinsic tryptophan (Trp-214). Besides the PT tautomer fluorescence (λmax = 526 nm), these protein–probe complexes exhibit a 3-HF anion emission (λmax = 500 nm). Analysis of spectroscopic data leads to the conclusion that two binding sites are involved in the human serum albumin–3-HF interaction. The 3-HF molecule bound in the higher affinity binding site, located in the IIIA subdomain, has the association constant (k1) of 7.2 × 105 M−1 and predominantly exists as an anion. The lower affinity site (k2 = 2.5 × 105 M−1), situated in the IIA subdomain, is occupied by the neutral form of 3-HF (normal tautomer). Since Trp-214 is situated in the immediate vicinity of the 3-HF normal tautomer bound in the IIA subdomain, the intermolecular energy transfer for this donor/acceptor pair has a 100% efficiency and is followed by the PT tautomer fluorescence. Intermolecular energy transfer from the Trp-214 to the 3-HF anion bound in the IIIA subdomain is less efficient and has the rate of 1.61 × 108 s−1, thus giving for the donor/acceptor distance a value of 25.5 Å.

Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study

Assembly and mutual proximities of α, β, and γc subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2Rα, IL-2Rβ, and γc subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2–10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2Rβ-α, γ-α, and γ-β pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a “triangular model” in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific α receptors with the β and/or γc subunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.

Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.