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At least 937 records · Page 52Linked to original sources
PubMed1999

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction.

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.

Filtration↗
PubMed1999

Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease.

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.

Amino Acid Sequence↗
PubMed2000

Red laser-induced fluorescence energy transfer in an immunosystem.

We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.

Antibodies↗
PubMed2002

A PDZ domain-based detection system for enzymatic assays.

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain.peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a "masked" biotinylated PDZ domain peptide ligand containing the consensus sequence Ser-X-Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu(3+) chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ) (GST-PDZ-glutathione S-transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase.

Carboxypeptidases↗
PubMed1997

Detection of DNA bending in a DNA-PAP1 protein complex by fluorescence resonance energy transfer.

The structure of DNA in a DNA-protein complex was studied by means of fluorescence resonance energy transfer (FRET) method. Oligonucleotide phosphorothioates were labeled with fluorescein and eosin to obtain a donor- and acceptor-labeled DNA. The formation of a complex of the DNA with PAP1(70), which is a DNA binding site fragment derived from transcription regulatory protein, PAP1, of fission yeast, was confirmed by gel retardation analysis and fluorescence measurements. FRET of the donor- and acceptor-labeled DNA with and without PAP1(70) indicated that the DNA in the complex was bent about 26 degrees toward the protein-binding surface.

Basic-Leucine Zipper Transcription Factors↗
PubMed2001

Molecular tryst peeping: detection of interactions between nonlabeled nucleic acids by fluorescence resonance energy transfer.

We have developed a new method for monitoring the interactions between nonlabeled RNAs that involves detection of fluorescence resonance energy transfer (FRET) between two DNA probes with different fluorescent label. The sequences of the probes are complementary to those of the RNAs. In this study, we examined the interaction between a portion of the LTR RNA of HIV-1 and the corresponding antisense RNA. The antisense RNA was designed not to bind to the fluorescent DNA without prior hybridization to the target RNA. A mixture of RNAs and DNA probes with fluorescent labels was fractionated by electrophoresis on a nondenaturing polyacrylamide gel and then the gel was analyzed with a fluorescence imaging analyzer. FRET was observed only in the presence of target RNA, antisense RNA, and both of the fluorescent DNA probes. This strategy should be useful for the detection of interactions between nucleic acids that cannot be subjected to chemical modification, such as RNA transcripts inside cells.

Base Sequence↗
PubMed1999

Determination of fluorescent probe orientations on biomolecules by conformational searching: algorithm testing and applications to the atomic model of myosin.

The ability of a localized conformational searching method to predict probe orientation was tested on model nucleic acid and protein structures and applied to the prediction of skeletal myosin integrity upon chemical modification of its reactive thiols. Double-stranded oligonucleotides were chemically labeled with donor and acceptor resonance energy transfer probes at each end for distance determinations. These measurements were made independently using a terbium chelate as a donor to each of four chemically and spectroscopically distinct acceptor probes from the xanthene and cyanine dye groups. The choice of acceptor significantly affected the separation distance measured. Conformational searching algorithms on the atomic model corrected for the differences to within 0.2 nm on average. Verifying its usefulness on proteins, the localized conformational searching method determined the orientation of a fluorescent probe on RNase A that corresponds closely to available crystallographic models of the labeled protein (RMS deviation = 0.1 nm). Also, analysis of the symmetry of the fluorophores' structures suggests why FRET orientation factors are often closer to their dynamic average value than might normally be expected. Furthermore, the computational method provides insights about FRET data that are important for assessing the stability of the alpha-helix separating the SH1 and SH2 reactive thiols in skeletal myosin.

Crystallography, X-Ray↗
PubMed2001

The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice.

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.

Animals↗
PubMed2000

Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.

When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.

Antigen Presentation↗
PubMed2000

Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins.

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.

Adenosine Diphosphate↗
PubMed1996

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer.

We have examined the fluorescence intensity decays of oxytocin and [Arg8]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu1,6-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (Rav) is similar in both compounds, Rav = 7.94 A for oxytocin and Rav = 8.00 A for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw = 2.80 A) than for oxytocin (hw = 3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking wih the Phe3 side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.

Amino Acid Sequence↗
PubMed1994

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ.

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.

Binding Sites↗
PubMed2005

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1.

We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.

Animals↗
PubMed2005

Detection of protein-protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer.

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)-protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein-protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP-protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP-protein A and EYFP-Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein-protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei.

Bacterial Proteins↗
PubMed2005

Intraspinal metalloma resulting in late paraparesis.

The metal-related complications caused by orthopedic implants have long been a concern, but these problems have been considered mostly in the field of arthroplasty or internal fixation of fractures. The recent prevalence of spinal instrumentation has evoked a similar concern among spine surgeons. Here, we present a case of intraspinal metallosis adjacent to the pedicular hook occurring after treatment of vertebral fracture by posterior spinal instrumentation and fusion, and causing paraparesis at the 3rd postoperative year. Metallic granulomas can appear around the pedicular hooks as in the reported case. Crevice and fretting corrosion are results at the junctions of rod-screw, rod-hook, transverse connector rod and other connector rods in modular spinal implants. Adequate usage of transpedicular screws may inhibit the occurrence of such a complication. For this reason, further studies are necessary to increase metallic corrosive resistance to inhibit crevice and fretting corrosion.

Biocompatible Materials↗
PubMed2005

Using co-cultures expressing fluorescence resonance energy transfer based protein biosensors to simultaneously image caspase-3 and Ca2+ signaling.

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca2+ signaling events involved in cell death of COS-7 cells induced by 10 mM H2O2. Interestingly, this H2O2 stimulus induced synchronous caspase-3 activation and Ca2+ signaling. In parallel to caspase-3 activation, cytosolic Ca2+ concentration, [Ca2+]c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca2+]c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H2O2. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.

Animals↗
PubMed2006

Voltage-dependent N-type Ca2+ channel activity regulates the interaction between FGF-1 and S100A13 for stress-induced non-vesicular release.

1. The Ca(2+)-mediated regulation of interaction between FGF-1 and S100A13 in NG108-15 cells was studied. When the stress by depriving B27 supplement from the culture was given, cellular levels of both proteins were decreased, while their releases were significantly increased within 3 h. These stress-induced changes were all abolished by amlexanox, an anti-allergic drug. 2. These releases were significantly inhibited by the addition of EGTA or BAPTA-AM, cellular or extracellular Ca(2+)-chelating agent, respectively. The addition of omega-conotoxin GVIA, a N-type Ca(2+)-channel blocker caused a complete inhibition of the release, while increased the cytosolic levels of both proteins. However, omega-conotoxin MVIIC, the non-N-type Ca(2+)-channel blocker was ineffective. 3. In NG108-15 cells, which had been transfected with Venus-FGF-1 and CFP-S100A13, the supplement-deprivation stress caused several spike-type fluorescence resonance energy transfer (FRET) signals, suggesting that both proteins showing interaction would be immediately released. These spikes were completely abolished by the addition of omega-conotoxin GVIA. However, the addition of amlexanox caused bell-shaped FRET signals without spikes. 4. Thus, it is suggested that the interaction between FGF-1 and S100A13 responsible for stress-induced non-vesicular release is dependent of Ca(2+)-influx through N-type Ca(2+)-channels.

Aminopyridines↗
PubMed2005

A new fluorescence resonance energy transfer pair and its application to oligonucleotide labeling and fluorescence resonance energy transfer hybridization studies.

We describe two new fluorescence resonance energy transfer (FRET) compatible labels, their covalent linkage to oligonucleotides, and their use as donor and acceptor, respectively, in FRET hybridization studies. The dyes belong to the cyanine dyes, and water solubility is imparted by a phosphonate which represents a new solubilizing group in DNA labels. They were linked to amino-modified synthetic oligonucleotides via oxysuccinimide (OSI) esters. The studies performed include binding assays, determinations of molecular distances, homogeneous competitive assays, and limits of detection, which are in the order of 5 pmol/L for a 15-mer.

Journal Article↗