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PubMed2006

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

DNA↗
PubMed2004

Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer.

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10(-6) m CORT than at 10(-9) m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

Animals↗
PubMed2005

Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis.

Fluorescence lifetime imaging microscopy presents a powerful tool in biology and biophysics because it allows the investigation of the local environment of a fluorochrome in living cells in a quantitative manner. Furthermore, imaging Förster-type resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy enables protein-protein interactions and intermolecular distances to be mapped under physiological conditions. Quantitative and precise data analysis methods are required to access the richness of information that is contained in FRET data on biological samples. Lifetime detection in the frequency-domain yields two lifetime estimations. The lifetime moments analysis (LiMA) provides a quantitative measure of the lifetime distribution broadness by exploiting the analytical relationship between the phase- and demodulation-lifetime estimations and relating them to the weighted average and variance of the lifetime distribution. The LiMA theoretical framework is validated by comparison with global analysis and by applying it to a constrained two-component FRET system using simulations and experiments. Furthermore, a novel LIMA-based error analysis and a more intuitive formalism for global analysis are presented. Finally, a new method to resolve a FRET system is proposed and experimentally applied to the investigation of protein-protein interactions.

Algorithms↗
PubMed2004

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations.

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.

Enzyme Activation↗
PubMed2001

Clustering of class I HLA oligomers with CD8 and TCR: three-dimensional models based on fluorescence resonance energy transfer and crystallographic data.

Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.

Antigen-Presenting Cells↗
PubMed2006

Evidence for ligand-independent multimerization of the IL-17 receptor.

IL-17 and its receptor are founding members of a novel inflammatory cytokine family. To date, only one IL-17 receptor subunit has been identified, termed IL-17RA. All known cytokine receptors consist of a complex of multiple subunits. Although IL-17-family cytokines exist as homodimers, the configuration and stoichiometry of the IL-17R complex remain unknown. We used fluorescence resonance energy transfer (FRET) to determine whether IL-17RA subunits multimerize, and, if so, whether they are preassembled in the plasma membrane. HEK293 cells coexpressing IL-17RA fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate FRET before and after IL-17A or IL-17F treatment. In the absence of ligand, IL-17RA molecules exhibited significant specific FRET efficiency, demonstrating that they exist in a multimeric, preformed receptor complex. Strikingly, treatment with IL-17A or IL-17F markedly reduced FRET efficiency, suggesting that IL-17RA subunits within the IL-17R complex undergo a conformational change upon ligand binding.

Animals↗
PubMed2003

Disruption of an intersubunit interaction underlies Ca2+-calmodulin modulation of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated channels are key molecular elements for olfactory transduction. Olfactory adaptation caused by repeated exposure to an odorant has been proposed to be mediated by the binding of Ca2+-calmodulin to the NH2-terminal domain of the channel, breaking its interaction with the COOH-terminal domain and downregulating the channel. We used a fluorescence resonance energy transfer (FRET) approach to study the structural aspects of this domain-domain interaction under physiological conditions in real time. Fluorescent proteins enhanced cyan fluorescent protein and enhanced yellow fluorescent protein were genetically attached at sites adjacent to the NH2- and COOH-terminal interacting domains, respectively, allowing direct observation of molecular rearrangements in intact channels. FRET signals caused by the specific interdomain interaction were observed in both intact cells and excised patches. Comparison of the effective FRET efficiencies demonstrated that the interaction occurs specifically between subunits but not within the same subunit. Binding of Ca2+-calmodulin caused a reversible decrease in FRET with the same time course as channel downregulation. These results suggest that a separation or reorientation of the interacting domains between subunits by Ca2+-calmodulin leads to channel downregulation. The quaternary arrangement presents a structural framework for understanding the molecular mechanism of olfactory adaptation.

Adaptation, Physiological↗
PubMed2003

Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.

Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.

Animals↗
PubMed2005

[Development and application of a new ADAMTS13 assay for TTP diagnosis].

A plasma glycoprotein, von Willebrand factor (VWF), is essential for normal platelet aggregation. In healthy individuals, the homo-multimeric forms (VWF multimers) are partially cleaved by a plasma metalloprotease, ADAMTS13. Congenital or acquired deficiency of ADAMTS13 activity leads to the accumulation of hyperactive large VWF multimers, inducing a life-threatening disease, thrombotic thrombocytopenic purpura (TTP). As measuring ADAMTS13 activity is important in TTP diagnosis, a number of assay methods have been developed in the past few years. However, the time and skill required for these methods prohibited the progress of clinical usage. Recently, we have developed a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. A synthetic 73-amino-acid peptide, FRETS-VWF73, which is now commercially available, is used as a substrate. Cleavage of this peptide between two modified residues relieves the fluorescence quenching in the intact form. Incubation of FRETS-VWF73 with normal plasma quantitatively increased fluorescence over time, while TTP-patient plasma had little or no effect. The measurement can be achieved within a one-hour period using a 96-well format in commercial plate readers with common filters. The FRET assay will be useful not only for TTP diagnosis but also characterization of thrombotic microangiopathies.

ADAM Proteins↗
PubMed2006

Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.

BACKGROUND AND AIM OF THE WORK: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis. METHODS: We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection. RESULTS: The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs. CONCLUSIONS: Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.

Adult↗
PubMed1991

Structural interpretation of fluorescence resonance-energy transfer measurements.

Fluorescence resonance-energy transfer (FRET) has been widely used to determine distance information in macromolecular systems. However, little has been written about methods for combining FRET distances into coherent structural models. I argue that the methods used so far are inappropriate. This paper describes an algorithm specifically tailored for finding structures from FRET measurements. This algorithm finds structures which fit the experimentally measured parameter, the efficiency of energy transfer, rather than derived distances. The algorithm was implemented in Mathematica and applied to FRET distances obtained for the contractile protein actin. The approach used is applicable to other experimental techniques which measure distances between a relatively small number of loci.

Energy Transfer↗
PubMed1994

Concerns with modularity in total hip arthroplasty.

Modularity is being diversified in total hip prostheses to increase surgical latitude in optimizing implant fixation and adjusting hip biomechanics. However, several problems have been clearly identified with implant modularity. First generation metal-backed acetabular components have shown deficiencies in the locking mechanism, the congruency and extent of polyethylene liner support, and polyethylene thickness, all of which have been implicated in accelerated polyethylene wear and failure. Evidence of screw motion against the metal backing, release of particulate material, and focal osteolysis have also been observed. At the head/neck junction evidence of corrosion and fretting has been documented with both similar-metal and mixed-metal taper combinations. Femoral prostheses with other sites of modularity present additional concerns with regard to mechanical integrity and generation of particulate debris by fretting. The modular junctions of three hip prostheses, the S-ROM, Infinity, and RMHS, were subjected to wet environment high cycle mechanical testing in a worst-case loading scenario. Preliminary results at relatively low loads up to three times body weight indicated gross stability of the modular junctions with evidence of minor fretting damage. Analysis of water solutions surrounding the modular junctions after ten to 20 million loading cycles yielded counts of one to three micron sized particles totalling several hundred thousand to several million. It is unknown what quantity of particulate material is sufficient to cause macrophage-mediated osteolysis or whether the debris from modular junctions can cause third-body wear of the articulating surfaces. Modular hip prostheses should be examined under stringent test conditions in order to characterize their fretting behavior and establish their mechanical limitations.

Biomechanical Phenomena↗
PubMed1998

Analysis of c-kit receptor dimerization by fluorescence resonance energy transfer.

Stem cell factor (SCF) binding to the c-kit receptor triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction. Receptor dimerization is a critical early step in this process. Prior biochemical studies of c-kit receptor dimerization have mainly used affinity cross-linking techniques, which are beset with problems including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label cytokine-responsive human hematopoietic cell lines. The ability of SCF to induce c-kit receptor dimerization was assessed by flow cytometric analysis of FRET between the donor fluorochrome FITC and the acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in c-kit receptor dimerization that correlated well with the concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF. Confocal microscopy showed redistribution of the c-kit receptor (from a diffuse distribution on the cell surface to "caps" at one end of the cell) within 3 minutes after SCF addition, followed by receptor internalization. Reappearance of the c-kit receptor on the cell surface required new protein synthesis, suggesting that the c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the structurally and functionally related cytokine thrombopoietin (Tpo), stimulated c-kit receptor dimerization detectable by FRET, and tyrosine phosphorylation of the c-kit receptor. These results suggest that exposure to Epo can activate the c-kit receptor and provide further evidence for cross-talk between the Epo and c-kit receptors in human hematopoietic cell lines. Studies with progeny of burst-forming unit-erythroid (BFU-E) suggest that the FRET technique is sufficiently sensitive to detect c-kit receptor dimerization on normal human hematopoietic cells.

Carbocyanines↗
PubMed1999

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E.

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Apolipoprotein E3↗
PubMed2001

Heterogeneity of second messenger levels in living cells.

Over the last years we have utilised chimeras from aequorin and green fluorescent protein (GFP) to monitor the dynamics of second messenger levels in living cells. In this contribution we address two problems, i.e. the complexity of Ca2+ handling by mitochondria and the localization of cAMP signalling. As to the first, we here demonstrate that physiological increases in mitochondrial Ca2+, monitored with selectively localized recombinant aequorin, concern a sub-population of organelles that is stably and selectively associated with the endoplasmic reticulum. As to cAMP, we describe the use of a novel probe to monitor its changes in living cells, that takes advantage of the phenomenon of fluorescence resonance energy transfer (FRET) between suitable GFPs linked to the regulatory and catalytic subunits of protein kinase A (PKA). When cAMP is low the two fluorophores are in close proximity and generate FRET while increasing levels of cAMP determine progressive reduction of FRET as the two subunits (linked to the GFPs) diffuse apart. We also demonstrate that by using such cAMP sensor, localized increase of this second messenger can be observed upon selective stimulation of plasma membrane receptors.

Animals↗
PubMed2006

Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations.

The use of Förster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.

Fluorescence Resonance Energy Transfer↗
PubMedDate not supplied

Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.

The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 A, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor-acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids.

Base Sequence↗
PubMed2004

Cytosine detection by a fluorescein-labeled probe containing base-discriminating fluorescent nucleobase.

We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base-discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ((ND)A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of (ND)A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of (ND)A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of (ND)A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400-500 nm. Thus, we designed FRET-BDF probes containing (ND)A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET-BDF probes was observed only when the complementary base of (ND)A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the (ND)A fluorescence by a G/C base pair flanking (ND)A.

Base Pairing↗