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At least 721 records · Page 40Linked to original sources
PubMed2006

A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.

Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.

Amino Acid Sequence↗
PubMed2006

Development and comparison of nonradioactive in vitro kinase assays for NIMA-related kinase 2.

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.

Animals↗
PubMed2006

Development of an open sandwich fluoroimmunoassay based on fluorescence resonance energy transfer.

We have developed a sensitive, one-step, homogeneous open sandwich fluoroimmunoassay (OsFIA) based on fluorescence resonance energy transfer (FRET) and luminescent semiconductor quantum dots (QDs). In this FRET assay, estrogen receptor beta (ER-beta) antigen was incubated with QD-labeled anti-ER-beta monoclonal antibody and Alexa Fluor (AF)-labeled anti-ER polyclonal antibody for 30 min, followed by FRET measurement. The dye separation distance was estimated between 80 and 90 A. The current method is rapid, simple, and highly sensitive, and it did not require the bound/free reagent separation steps and solid-phase carriers. A concentration as low as 0.05 nM (2.65 ng/ml) receptor was detected with linearity. In addition, the assay was performed with commercial antibodies. This assay provides a convenient alternative to conventional, laborious sandwich immunoassays.

Antibodies, Monoclonal↗
PubMed2006

Subunit movements in membrane-integrated EF0F1 during ATP synthesis detected by single-molecule spectroscopy.

The H+ -ATPsynthase from E. coli was isolated and labelled at the gamma- or epsilon-subunit with tetramethylrhodamine, and at the b-subunits with bisCy5. The double labelled enzymes were incorporated into liposomes. They showed ATP hydrolysis activity, and, after energization of the membrane by DeltapH and Deltavarphi, also ATP synthesis activity was observed. Fluorescence resonance energy transfer (FRET) was used to investigate the movements of either the gamma-subunit or the epsilon-subunit relative to the b-subunits in single membrane-integrated enzymes. The results show that during catalysis, the gamma-epsilon complex rotates stepwise relative to the b-subunit. The direction of rotation during ATP synthesis is opposite to that during ATP hydrolysis. The stepwise motion is characterized by dwell times (docking time of the gamma-epsilon complex to one alphabeta pair) up to several hundred ms, followed by a rapid movement of the gamma- and epsilon-subunit to the next alphabeta pair within 0.2 ms. The same FRET levels (i.e., the same gamma-b and epsilon-b distances) are observed during proton transport-coupled ATP hydrolysis and ATP synthesis, indicating that the reaction proceeds via the same intermediates in both directions. Under non-catalytic conditions, i.e., in the absence of ATP or without energization also, three FRET levels are found, however, the distances differ from those under catalytic conditions. We conclude that this reflects a movement of the epsilon-subunit during active/inactive transition.

Adenosine Triphosphate↗
PubMed2005

Use of Forster's resonance energy transfer microscopy to study lipid rafts.

Rafts in cell membranes have been a subject of much debate and many models have been proposed for their existence and functional significance. Recent studies using Forster's resonance energy transfer (FRET) microscopy have provided one of the first glimpses into the organization of putative raft components in living cell membranes. Here we discuss how and why FRET microscopy provides an appropriate non-invasive methodology to examine organization of raft components in cell membranes; a combination of homo and hetero-FRET microscopy in conjunction with detailed theoretical analyses are necessary for characterizing structures at nanometre scales. Implications of the physical characteristics of the organization of GPI-anchored proteins in cell membranes suggest new models of lipid-based assemblies in cell membranes based on active principles.

Animals↗
PubMed2006

gp130 dimerization in the absence of ligand: preformed cytokine receptor complexes.

It is established that cytokine receptors signal after ligand binding as homo- or hetero-dimers in heteromeric complexes, but it is unclear, when dimerization occurs. To investigate gp130 dimerization, we performed co-precipitation experiments with the endogenous cytokine receptors gp130 and leukemia inhibitory factor receptor (LIF-R) and with gp130 variants carrying two different C-terminal peptide tags. Furthermore, fluorescence resonance energy transfer (FRET) was employed to detect dimerization of two fluorescent-tagged gp130 variants. Confocal laser scanning microscopy was used for FRET detection in live cells. gp130 and LIF-R could be coprecipitated in the absence of ligand. The interaction, however, was intensified by the addition of LIF. Similar results were obtained with the gp130 variants and confirmed by FRET analysis in live cells. The present study clearly demonstrates the existence of preformed but inactive gp130/LIF-R hetero- and gp130/gp130 homo-dimers. The addition of ligand enhanced the respective dimer formation and was required for signal transduction.

Animals↗
PubMed2006

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

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

Animals↗
PubMed2005

Single molecule fluorescence studies of surface-adsorbed fibronectin.

The conformation of the extracellular matrix protein fibronectin plays a critical role in regulating cell function, including cell adhesion and migration. While average conformations of large ensembles of adhesion proteins have been previously measured, cells may sensitively respond to conformational outliers. We therefore applied both single molecule imaging and spectroscopy techniques to map a range of conformational states of individual fibronectin molecules adsorbed to glass, as well as to measure their conformational fluctuations in time. Single-step photobleaching experiments confirmed single molecule sensitivity. Single molecule spectra showed fluctuations in the peak wavelength, both over a spatial ensemble of molecules and in a single molecule over time, most likely indicating the different conformational states fibronectin can assume upon surface adsorption. Single-pair fluorescence resonance energy transfer (FRET) revealed that a fraction of fibronectin molecules existed in conformations that allowed for energy transfer between the labeled cysteine residues of the two dimeric arms folded upon each other, and that fluctuations occurred in the FRET efficiency. Fluorescence polarization experiments identified two possible sources of FRET fluctuations: changes in fluorophore orientation and conformational fluctuations of fibronectin over a time scale of seconds.

Adsorption↗
PubMed2004

Synthesis, biophysical and biological evaluation of 3,6-bis-amidoacridines with extended 9-anilino substituents as potent G-quadruplex-binding telomerase inhibitors.

Telomerase and telomere maintenance are emerging targets for the treatment of human cancers. We report here on the targeting of the telomere-telomerase complex with a series of small molecules based on an acridine platform. A series of 3,6-bisamidoacridines with extended 9-anilino sidechains were designed and synthesised as potential telomeric G-quadruplex DNA (G4) interacting compounds. G4-stabilisation was assessed using a high-throughput FRET (fluorescence resonance energy transfer) assay and telomerase inhibition quantified by a modified TRAP (telomerase repeat amplification protocol) method. Within the series, the compounds showed significant G4-stabilising ability (Delta T(m) values of 25-36 degrees C at 1 microM concentration) and telomerase inhibition in the nanomolar region ((tel)EC(50) values of 80-318 nM). Furthermore, a direct correlation between the FRET and TRAP assays was observed, supporting the use of the rapid screening FRET assay for early assessment of potential G4-stabilising telomerase inhibitors.

Acridines↗
PubMed2005

Quantitative determination of the topological propensities of amyloidogenic peptides.

One of the interesting puzzles of amyloid beta-peptide of Alzheimer's disease (Abeta) is that it appears to polymerize into amyloid fibrils in a parallel beta sheet topology, while smaller subsets of the peptide produce anti-parallel beta sheets. In order to target potential weak points of amyloid fibrils in a rational drug design effort, it would be helpful to understand the forces that drive this change. We have designed two peptides CHQKLVFFAEDYNGKDEAFFVLKQHW and CHQKLVFFAEDYNGKHQKLVFFAEDW that join the significant amyloidogenic Abeta (14-23) sequence HQKLVFFAED in parallel and anti-parallel topologies, respectively. (Here, the word "parallel" refers only to residue sequence and not backbone topology). The N-termini of the hairpins were labeled with the fluorescent dye 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), forming a fluorescence energy transfer donor-acceptor pair with the C-terminus tryptophan. Circular dichroism results show that the anti-parallel hairpin adopts a beta-sheet conformation, while the parallel hairpin is disordered. Fluorescent Resonance Energy Transfer (FRET) results show that the distance between the donor and the acceptor is significantly shorter in the anti-parallel topology than in the parallel topology. The fluorescence intensity of anti-parallel hairpin also displays a linear concentration dependence, indicating that the FRET observed in the anti-parallel hairpin is from intra-molecular interactions. The results thus provide a quantitative estimate of the relative topological propensities of amyloidogenic peptides. Our FRET and CD results show that beta sheets involving the essential Abeta (14-23) fragment, strongly prefer the anti-parallel topology. Moreover, we provide a quantitative estimate of the relative preference for these two topologies. Such analysis can be repeated for larger subsets of Abeta to determine quantitatively the relative degree of preference for parallel/anti-parallel topologies in given fragments of Abeta.

Amino Acid Sequence↗
PubMed2004

Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers.

Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.

Biopolymers↗
PubMed2005

Coupling of size-exclusion chromatography to a continuous assay for subtilisin using a fluorescence resonance energy transfer peptide substrate: testing of two standard inhibitors.

Liquid chromatography (LC) was coupled on-line to a homogeneous continuous-flow protease assay using fluorescence resonance energy transfer (FRET) as a readout for the screening of inhibitors of an enzyme (e.g., Subtilisin Carlsberg). The inhibitors aprotinin (a protein of approximately 6500 g/mol) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 240 g/mol) were mixed with other, non-active compounds and separated on a size-exclusion chromatography column. After the separation, the analytes were eluted to the postcolumn reactor unit where the enzyme solution and subsequently the FRET peptide substrate were added; by measuring the fluorescence intensity the degree of inhibition was monitored on-line. As expected, only the two inhibitors caused a change in the FRET response. Detection limits for aprotinin were 5.8 microM in the flow injection analysis (FIA) mode and 12 microM in the on-line LC mode. System validation was performed by determining IC50 values for aprotinin for the FIA mode (19 microM) and the on-line mode (22 microM). These IC50 values were in line with the value determined in batch experiments (25 microM). With this system, chemical information (i.e., chromatographic retention time) and biological information (i.e., enzyme inhibition) can be combined to characterize mixtures.

Aprotinin↗
PubMed2006

Maltose-binding protein: a versatile platform for prototyping biosensing.

The bacterial periplasmic-binding protein (PBP) superfamily members, in particular the maltose-binding protein, have been used extensively to prototype a variety of biosensing platforms. Although quite diverse at the primary sequence level, this protein superfamily retains the same basic two-domain structure, and upon binding a recognized ligand almost all PBPs undergo a conformational change to a closed structure. This process forms the basis for most, but not all, PBP-based biosensor signal transduction. Many direct detection or reagentless sensing modalities have been utilized with maltose-binding protein for both in vitro and in vivo detection of target compounds. Signal transduction modalities developed to date include direct fluorescence, electrochemical detection, fluorescence resonance energy transfer (FRET)-based detection, surface-tethered FRET sensing, hybrid quantum dot FRET sensing, and enzymatic detection, each of which have different benefits, potential applications and limitations.

Biosensing Techniques↗
PubMed2006

Fluorescence resonance energy transfer: evaluation of the intracellular stability of polyplexes.

The investigation of intracellular mechanisms of non-viral nucleic acid delivery systems has provided great impetus for the improvement of their efficacy. Especially the intracellular release of the nucleic acid from the non-viral carrier system may be a relevant criterion for the high transfection efficiency of certain polymers. Therefore, we evaluated fluorescence resonance energy transfer (FRET) in combination with confocal laser scanning microscopy or flow cytometry as tool to determine the intracellular disintegration of polyplexes built with plasmid DNA and linear polyethylenimine. In microscopy, which allowed for an observation of polyplexes within single cells, sensitized emission measurement and acceptor photobleaching have been tested towards quantitative FRET analysis. In contrast, the whole cell population was analyzed by the flow cytometry-based method. We suggest that FRET is a useful tool to evaluate the intracellular disintegration of polyplexes built with various polymers.

Animals↗
PubMed2006

A novel analytical method for in vivo phosphate tracking.

Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.

Animals↗
PubMed2004

Nitric oxide-induced modification of protein thiolate clusters as determined by spectral fluorescence resonance energy transfer in live endothelial cells.

Low-molecular-weight S-nitrosothiols are found in many tissues and affect a diverse array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of D- and L-stereoisomers of S-nitrosocysteine ethyl ester (SNCEE) on fluorescence resonance energy transfer (FRET)-based reporters that are targets for the following NO-related modifications: (a) S-nitrosation, via the cysteine-rich protein metallothionein (FRET-MT), and (b) nitrosyl-heme-Fe, via guanosine 3',5'-cyclic monophosphate (cygnet-2). Conformational changes consistent with S-nitrosation of FRET-MT were specific to l-SNCEE. In addition, they were reversed by dithiothreitol (DTT) but unaffected by exogenous oxyhemoglobin. In contrast, d- and l-SNCEE had comparable effects on cygnet-2, likely via activation of soluble guanylyl cyclase (sGC) by *NO as they were sensitive to the sGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-alpha] quinoxalin-1-one and exogenous oxyhemoglobin. These data demonstrate the utility of spectral laser scanning confocal imaging in revealing subtle aspects of NO signal transduction in live cells. Stereoselective transnitrosation of MT emphasizes the specificity of posttranslational modification as a component of NO signaling.

Animals↗
PubMed2005

Clear evidence of fluorescence resonance energy transfer in gas-phase ions.

Fluorescence resonance energy transfer (FRET) is a distance-sensitive method that correlates changes in fluorescence intensity with conformational changes, for example, of biomolecules in the cellular environment. Applied to the gas phase in combination with Fourier transform ion cyclotron resonance mass spectrometry, it opens up possibilities to define structural/conformational properties of molecular ions, in the absence of solvent, and without the need for purification of the sample. For successfully observing FRET in the gas phase it is important to find suitable fluorophores. In this study several fluorescent dyes were examined, and the correlation between solution-phase and gas-phase fluorescence data were studied. For the first time, FRET in the gas phase is demonstrated unambiguously.

Fluorescence Resonance Energy Transfer↗
PubMed2005

A rapid real-time qRT-PCR assay for ovine beta-actin mRNA.

Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells.

Actins↗