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At least 289 records · Page 16Linked to original sources
PubMed2002

An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction.

An economical and preparative-scale orthogonal solid-phase method of incorporating carboxyrhodamine and carboxyfluorescein fluorescence resonance energy transfer (FRET) probes site-specifically into synthetic peptide substrates for the S. aureus Sortase transpeptidase SrtA has been developed.

Aminoacyltransferases↗
PubMed2006

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

Dinitrobenzenes↗
PubMed2006

Regulation of RalA GTPase by phosphatidylinositol 3-kinase as visualized by FRET probes.

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

Cell Membrane↗
PubMed2001

Combining AFM and FRET for high resolution fluorescence microscopy.

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.

Journal Article↗
PubMed2004

Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system.

Two-component signaling systems, in which a receptor-coupled kinase is used to control the phosphorylation level of a response regulator, are commonly used in bacteria to sense their environment. In the chemotaxis system of Escherichia coli, the receptors, and thus the kinase, are clustered on the inner cell membrane. The phosphatase of this system also is recruited to receptor clusters, but the reason for this association is not clear. By using FRET imaging of single cells, we show that in vivo the phosphatase activity is substantially larger at the cluster, indicating that the signaling source (the kinase) and the signaling sink (the phosphatase) tend to be located at the same place in the cell. When this association is disrupted, a gradient in the concentration of the phosphorylated response regulator appears, and the chemotactic response is degraded. Such colocalization is inevitable in systems in which the activity of the kinase and the phosphatase are produced by the same enzyme. Evidently, this design enables a more rapid and spatially uniform response.

Bacterial Proteins↗
PubMed2005

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation.

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.

Bacterial Toxins↗
PubMed2005

Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes.

Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.

Bacteriophage lambda↗
PubMed2003

Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.

The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.

Animals↗
PubMed2006

Development and evaluation of a real-time FRET probe based multiplex PCR assay for the detection of prohibited meat and bone meal in cattle feed and feed ingredients.

A novel real-time fluorescent multiplex polymerase chain reaction (PCR) assay for detecting and discriminating between bovine, ovine, and caprine contaminates in cattle feed was developed that simultaneously performs quality control monitoring on both the DNA extraction process and the level of PCR inhibition in the final DNA extract in a single PCR run. The assay used a single set of primers and two sets of FRET probes targeting the ruminant-specific mitochondrial cytochrome b gene. An internal control PCR reaction targeting a region of the chloroplast RNA polymerase beta-subunit (rpobeta) gene, which is conserved among plants, was incorporated into the ruminant multiplex PCR reaction in order to both monitor the DNA extraction method and to test for the presence of PCR inhibitors. The detection limit for bovine and ovine contaminates was evaluated over a period of two sets of six trials on 15 different types of cattle feed and feed ingredients spiked with known concentrations of bovine meat and bone meal (BMBM) and lamb meat and bone meal (LMBM). The assay was able to detect 0.05% w/w BMBM contamination and 0.1% w/w LMBM contamination in all samples of cattle feed and feed ingredients tested.

Animal Feed↗
PubMed2005

Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system.

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.

Animals↗
PubMed2004

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.

Alleles↗
PubMed2005

Complexation of thrombin-binding aptamer oligonucleotide carrying fluorescence resonance energy transfer (FRET) dyes at both termini with potassium ion.

Oligonucleotide derivatives, F-TBA-T and F-7TBA-T carrying thrombin-binding aptamer (TBA) sequence and FAM and TAMRA dyes at their each end were designed to achieve the fluorometric monitoring of potassium ion (K+) concentrations in a living organisms. These oligonucleotides could form a tetraplex structure upon addition of K+. Spectrophotometric experiments revealed that the fluorescence enhancement on the fluorescence resonance energy transfer (FRET) was larger in the case of F-7TBA-T having 7-nucleotides at 5'-end of TBA sequence than in the case of F-TBA-T due to the elimination of the quenching by dye-dye interaction.

Aptamers, Nucleotide↗
PubMed2005

Affinity and selectivity of G4 ligands measured by FRET.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the stabilization and selectivity of two well-known quadruplex ligands (telomestatin and a cationic porphyrin) towards the human telomeric G-quadruplex species, with FRET. Both molecules strongly stabilize the G-quadruplex, but telomestatin appears much more selective, as shown by competition experiments with double-stranded DNA.

DNA↗
PubMed1999

Why FRET over genomics?

Genetic information is being uncovered quickly and in vast amounts through the largely automated sequencing of genomes from all kinds of organisms. As this information becomes available, enormous challenges are emerging on three levels: first, functions will have to be assigned to individual gene products; second, factors that influence the expression level of these gene products will have to be identified; and third, allelic variants that act alone or in combination to give rise to complex traits will have to be characterized. Because of the sheer size of genomes, methods that can streamline or automate these processes are highly desirable. Fluorescence is an attractive readout for such high-throughput tasks because of the availability of equipment designed to detect light-emitting compounds with great speed and high capacity. The following is an overview of the achievements and potential of fluorescence resonance energy transfer (FRET) as applied in three areas of genomics: the identification of single-nucleotide polymorphisms, the detection of protein-protein interactions, and the genomewide analysis of regulatory sequences.

Animals↗
PubMed2006

Assay concordance between SPA and TR-FRET in high-throughput screening.

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.

Biological Assay↗
PubMed2005

Study of the pH-dependent conformational changes in alpha1-acid glycoprotein using FRET.

We studied pH-dependent conformational changes in alpha1-acid glycoprotein using both binding of cationic fluorescent probe Quinaldine Red with protein molecule and changes in distance between tryptophan from binding site of the protein and Calcofluor White on glycan surface using FRET. Our data show that the binding site of the protein has undergone a conformational change at pH 9.0. At this pH. the binding site forms a more hydrophobic region and tryptophan residues from the binding site move to the center of the protein core.

Benzenesulfonates↗