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At least 217 records · Page 12Linked to original sources
PubMed2006

Structural analysis of the catalytic core of human telomerase RNA by FRET and molecular modeling.

Telomerase is the ribonucleoprotein reverse transcriptase involved in the maintenance of the telomeres, the termini of eukaryotic chromosomes. The RNA component of human telomerase (hTR) consists of 451 nucleotides with the 5' half folding into a highly conserved catalytic core comprising the template region and an adjacent pseudoknot domain (nucleotides 1-208). While the secondary structure of hTR is established, there is little understanding of its three-dimensional (3D) architecture. Here, we have used fluorescence resonance energy transfer (FRET) between fluorescently labelled peptide nucleic acids, hybridized to defined single stranded regions of full length hTR, to evaluate long-range distances. Using molecular modeling, the distance constraints derived by FRET were subsequently used, together with the known secondary structure, to generate a 3D model of the catalytic core of hTR. An overlay of a large set of models generated has provided a low-resolution structure (6.5-8.0 A) that can readily be refined as new structural information becomes available. A notable feature of the modeled structure is the positioning of the template adjacent to the pseudoknot, which brings a number of conserved nucleotides close in space.

Catalytic Domain↗
PubMed1998

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA.

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.

Adenosine Triphosphate↗
PubMed2002

A FRET-based ultraviolet to near-infrared frequency converter.

The synthesis and photophysical characterization of a generation 1 dendrimer containing four coumarin 2 laser dyes at the periphery and a perylenebis(dicarboximide) derivative at the core are described. It was found that 99% of the UV light absorbed by the peripheral coumarin 2 donor chromophores is transferred through fluorescence resonance energy transfer (FRET) to the core acceptor. Emission from the core via FRET is observed in the near-infrared with a 6.2-fold amplification relative to direct excitation.

Journal Article↗
PubMed2006

A dual parameter FRET probe for measuring PKC and PKA activity in living cells.

Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.

Blood Proteins↗
PubMed2006

FRET templated by G-quadruplex DNA: a specific ternary interaction using an original pair of donor/acceptor partners.

G-quadruplex represents a suitable scaffold for FRET (fluorescence resonance energy transfer) since its two external quartets offer two well-defined binding sites for concomitant trapping of donor/acceptor partners. Combining selective G-quadruplex binders (macrocyclic bis(quinacridine) BOQ(1) or monomeric quinacridine MMQ(1), donor) with a highly fluorescent DNA probe (thiazole orange, acceptor), we designed a structure-specific FRET-system based on an unprecedented noncovalent ternary complex. This system could be potentially usable as a signature for quadruplex-DNA conformation in solution, but also might offer a unique means for observing cation and ligand binding influence on quadruplex topology.

Acridines↗
PubMed2006

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin.

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.

Biosensing Techniques↗
PubMed2006

Single-pair FRET characterization of DNA tweezers.

The self-assembly properties of DNA make it an ideal choice for the construction of nanomachines such as DNA tweezers. Because nanomachines function as individual units, they need to be studied on the single-molecule level. From single-pair FRET investigations, we show that "open" tweezers exist in a single conformation with minimal FRET efficiency, whereas upon addition of a "closing strand", three conformations are observed, which are averaged out in ensemble measurements.

DNA↗
PubMedDate not supplied

Implications of orthopedic fretting corrosion particles on skeletal muscle microcirculation.

Particulate corrosion and wear products of metal implants are increasingly becoming topics of interest, due to the cascade of biological and biomechanical events they induce. The impairment of skeletal muscle microcirculation by fretting corrosion particles may have profound consequences. We therefore studied in vivo leukocyte-endothelial cell interaction in skeletal muscle after confrontation with characterized titanium and stainless steel fretting corrosion particles, and compared these results with those of the bulk materials. Using the hamster dorsal skinfold chamber preparation and intravital microscopy, we could demonstrate that stainless steel induces a more pronounced inflammatory answer in contrast to the implant material titanium. However, we were not able to show a general benefit of bulk vs. debris. Overall, the study suggests that not only the bulk properties of orthopaedic implants, but also the microcirculatory implications of inevitable wear debris, may play a role in determining biocompatibility and ultimately longevity of an implant. The skinfold chamber is a feasible and versatile model for observation of the dynamic process of microvascular response after foreign-body implantation, and offers much perspective.

Journal Article↗
PubMed2004

FRET studies of the interaction of dimeric cyanine dyes with DNA.

Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10.

Carbocyanines↗
PubMed2004

Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns.

A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.

Amino Acid Sequence↗
PubMed1999

High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR).

We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.

DNA Primers↗
PubMed2003

FRET imaging.

Förster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.

Fluorescence Resonance Energy Transfer↗
PubMed2003

Self-assembled nanoscale biosensors based on quantum dot FRET donors.

The potential of luminescent semiconductor quantum dots (QDs) to enable development of hybrid inorganic-bioreceptor sensing materials has remained largely unrealized. We report the design, formation and testing of QD-protein assemblies that function as chemical sensors. In these assemblies, multiple copies of Escherichia coli maltose-binding protein (MBP) coordinate to each QD by a C-terminal oligohistidine segment and function as sugar receptors. Sensors are self-assembled in solution in a controllable manner. In one configuration, a beta-cyclodextrin-QSY9 dark quencher conjugate bound in the MBP saccharide binding site results in fluorescence resonance energy-transfer (FRET) quenching of QD photoluminescence. Added maltose displaces the beta-cyclodextrin-QSY9, and QD photoluminescence increases in a systematic manner. A second maltose sensor assembly consists of QDs coupled with Cy3-labelled MBP bound to beta-cyclodextrin-Cy3.5. In this case, the QD donor drives sensor function through a two-step FRET mechanism that overcomes inherent QD donor-acceptor distance limitations. Quantum dot-biomolecule assemblies constructed using these methods may facilitate development of new hybrid sensing materials.

Biosensing Techniques↗
PubMed2001

FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 microM.

Animals↗
PubMed2005

FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

3T3 Cells↗
PubMed2005

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.

Base Sequence↗
PubMed2006

Zinc binding to a regulatory zinc-sensing domain monitored in vivo by using FRET.

We have generated probes of metal binding to zinc fingers (ZFs) that provide tools to study zinc trafficking in vivo. In this study, we used these probes to examine zinc binding by the Zap1 transcription factor of Saccharomyces cerevisiae. Zap1 contains two zinc-regulated activation domains (ADs), AD1 and AD2. AD2 is located within two C2H2 ZFs, ZF1 and ZF2. Studies have indicated that apoAD2 activates transcription and zinc binding to ZF1 and that ZF2 forms an interacting-finger-pair structure that is necessary to inhibit AD function. A related structural finger pair, ZF3 and ZF4, is found in the Zap1 DNA binding domain. In vitro studies indicated that, although the ZF1/2 and ZF3/4 finger pairs bind zinc with similar affinities, zinc that was bound to ZF1/2 was much more labile. We examined the properties of Zap1 ZFs in vivo by FRET. ZF pairs were flanked by enhanced yellow fluorescent protein and enhanced cyan fluorescent protein, allowing detection of zinc-induced conformation changes by FRET. By using these reporters, we found that ZF1/2 and ZF3/4 showed similar responses to zinc under steady-state conditions in vivo. In contrast, ZF1/2 zinc binding was significantly more labile than was ZF3/4. Also, ZF1/2 accumulated in an apo form that could rapidly bind zinc, whereas the ZF3/4 pair did not. Last, we show that these properties are evolutionarily conserved indicating their importance to Zap1 function. These results indicate that the kinetic lability of ZF1/2 in vivo is a key component of Zap1 zinc responsiveness.

Amino Acid Sequence↗
PubMed2006

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.

Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.

Boron Compounds↗