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At least 163 records · Page 9Linked to original sources
PubMed2004

Fluorescein provides a resonance gate for FRET from conjugated polymers to DNA intercalated dyes.

Fluorescence spectra show that excitation of the cationic water-soluble conjugated polymer poly[(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethylammonium)-hexyl]fluorene diiodide] (1) results in inefficient fluorescence resonance energy transfer (FRET) to ethidium bromide (EB) intercalated within double-stranded DNA (dsDNA). When fluorescein (Fl) is attached to one terminus of the dsDNA, there is efficient FRET from 1 through Fl to EB. The cascading energy-transfer process was examined mechanistically via fluorescence decay kinetics and fluorescence anisotropy measurements. These experiments show that the proximity and conformational freedom of Fl provide a FRET gate to dyes intercalated within DNA which are optically amplified by the properties of the conjugated polymer. The overall process provides a substantial improvement over previous homogeneous conjugated polymer based DNA sensors, namely, in the form of improved selectivity.

Base Sequence↗
PubMed2004

Genetically encoded FRET probe for PKC activity based on pleckstrin.

We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.

Bacterial Proteins↗
PubMed2006

Synthesis and application of quantum dots FRET-based protease sensors.

Preparation of FRET-based quantum dots as protease sensors-RGDC peptide molecules are bound to the surface of CdSe/ZnS quantum dots. The peptide molecules are then labeled with rhodamine dye molecules. The emission color of the quantum dots change from green to orange due to fluorescence resonance energy transfer (FRET) between the quantum dots and the bound rhodamine molecules. Cleavage of the peptide by selective proteases releases the rhodamine molecules from the quantum dots surface, which results in decreasing FRET efficiency between the quantum dots and the rhodamine molecules. The emission color of the quantum dots changes back to green.

Enzyme Activation↗
PubMed2005

On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.

The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.

Benzenesulfonates↗
PubMed2006

Multicolor FRET silica nanoparticles by single wavelength excitation.

Fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stöber synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size, and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift, which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems.

Journal Article↗
PubMed2006

FRET-CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections.

It is pivotal to identify protein-protein interaction in situ to understand protein function. Conventional methods to determine the interaction of proteins destruct tissue or are applicable to cell culture only. To identify association of proteins in cells in tissue, we adapted indirect double-labeling immunofluorescence and combined it with conventional confocal laser scanning microscopy (CLSM) to measure fluorescence resonance energy transfer (FRET). As a model system, we chose caveolin-1alpha and caveolin-2, two major components of endothelial caveolae, and examined their interaction in the endothelium of vessels in fixed tissues of laboratory animals and human glomus tumors. Several methodological aspects were examined. Measuring the absolute increase in fluorescence (DeltaIF) was superior compared to determining the relative FRET efficiency, because it is more robust against small increases of fluorescence during measurements that results from unavoidable minimal crossreactivity of the secondary antibodies. Both, sequential and simultaneous incubation of secondary antibodies result in robust and reliable increases in DeltaIF. If incubated sequentially, however, the acceptor-labeled secondary antibody should be applied first. The size of the secondary reagent (F(ab')2 vs whole antibody) has no major influence. In conclusion, CLSM-FRET can measure close spatial association of proteins in situ and can be applied to human surgical material.

Animals↗
PubMed2004

An improved cyan fluorescent protein variant useful for FRET.

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Animals↗
PubMed2006

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.

Calmodulin↗
PubMed2007

A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine.

BACKGROUND AND PURPOSE: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. EXPERIMENTAL APPROACH: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called 'caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. KEY RESULTS: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. CONCLUSIONS AND IMPLICATIONS: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery.

Antineoplastic Agents↗
PubMed2002

Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules.

Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.

Energy Transfer↗
PubMed2005

Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes.

We describe the use of modified fluorescent-labeled oligonucleotide probes in the sequence-specific detection of messenger RNAs in live human cells. To make this detection possible, we developed a previously undescribed probe design that combines earlier quenched autoligation chemistry with a previously undescribed fluorescence resonance energy transfer (FRET) strategy to lower background signals. The probe pairs consisted of a nucleophilic 3'-phosphorothioate probe carrying a Cy5 FRET acceptor, and an electrophilic probe containing the combination of a 5' end electrophile/quencher and a fluorescein FRET donor. Probes were introduced to HL-60 cells by use of the streptolysin O pore-forming peptide. Signals from three different messenger RNAs, as well as 28S ribosomal RNA, could be detected and quantitated by flow cytometry. Probes targeted to ribosomal sequences and beta-actin mRNA also could be detected over background by confocal fluorescence microscopy. Varying the target site and probe backbone chemistry were found to have large effects on signal. The data suggest that quenched autoligating probes may be of general utility as biological tools in following localization, transcription, and processing of eukaryotic cellular messages and may have applications in diagnostic or prognostic analysis of disease-related RNAs in human tissues.

Flow Cytometry↗
PubMed2005

Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints.

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.

Capsid Proteins↗
PubMed2003

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis.

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.

Bacterial Proteins↗
PubMed2004

Analysing the action of bacterial toxins in living cells with fluorescence resonance energy transfer (FRET).

Bacterial toxins represent small molecules produced by microorganisms. Different toxins act on specific target molecules in mammalian cells. Once discovered, bacterial toxins have been providing tools to study cellular functions and often helped the dissection of complex cellular pathways, e.g. endocytic or secretory trafficking or signal transduction, by virtue of the fact that they either block or activate their specific cellular target molecules. Purified bacterial toxins have also allowed to address many basic biological questions and have provided tools for in vitro and in vivo experimental approaches in many fields of modern biology. The understanding of how bacterial toxins act in living cells often depends on our ability to visualize the trafficking and signaling pathways of these molecules. Fluorescence microscopy and other imaging tools are essential to provide insights into the functional changes induced by these pathogens at the level of individual host cells or single target proteins. Inside a single cell we can measure and quantify the effects of bacterial toxins on specific cellular proteins by microscopic and spectroscopic techniques. Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows to study protein-protein interactions. FRET can provide distance information in the range of 3- 7 nm between fluorescently labeled bacterial proteins in the live cell and cellular target proteins expressed as chimeras with green fluorescent protein (GFP), or spectrally shifted variants thereof. The purpose of this review is to introduce readers to the main experimental setups for analyses of protein-protein interactions using FRET as well as some applications.

Cholera Toxin↗
PubMed2002

Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy.

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

A Kinase Anchor Proteins↗
PubMed2003

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Adenoviridae↗
PubMed1994

Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET).

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.

Animals↗
PubMed2005

Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein.

Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the alpha-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ([[(amino)ethyl]amino]naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the alpha-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.

Biosensing Techniques↗