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At least 253 records · Page 14Linked to original sources
PubMed2002

Core-substituted naphthalene bisimides: new fluorophors with tunable emission wavelength for FRET studies.

Highly colored and photoluminescent naphthalene bisimide dyes have been synthesized from 2,6-dichloronaphthalene bisanhydride 1 by means of a stepwise nucleophilic displacement of the two chlorine atoms by alkoxides and/or alkyl amines. The alkoxy-substituted derivatives are yellow dyes with green emission and low photoluminescence quantum yields, whereas the amine-substituted derivatives exhibit a color range from red to blue with strong photoluminescence up to 76%. Structure-property relationships for this class of two-dimensional chromophores were evaluated based on a single-crystal X-ray analysis for dye 5a, the observed solvatochromism, and quantum-chemical calculations. Owing to the simple tuning of the absorption properties over the whole visible range by the respective substituents, the pronounced brilliancy, and the intense photoluminescence, this class of dyes is considered to be highly suited for numerous applications such as fluorescent labeling of biomacromolecules and light-harvesting in supramolecular assemblies. As an important step towards such applications efficient FRET (fluorescence resonance energy transfer) has been demonstrated for a covalently tethered bichromophoric compound that contains a red and a blue naphthalene bisimide dye.

Journal Article↗
PubMed2005

The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements.

The folding and dynamics of globular proteins is a multidimensional problem. The structures of the heterogeneous population of refolding protein molecules are characterized by multiple distances and time constants. Deciphering the mechanism of folding depends on studies of the processes rather than the folded structures alone. Spectroscopy is indispensable for these sorts of studies. Herein, it is shown that the determination of intramolecular distance distributions by ensemble and single-molecule FRET experiments enable the exploration of partially folded states of refolding protein molecules.

Adenylate Kinase↗
PubMed2004

Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells.

In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs).

Anisotropy↗
PubMed2001

Novel immuno-FRET assay method for Bacillus spores and Escherichia coli O157:H7.

Novel immunofluorescence resonance energy transfer (immuno-FRET) assays for both Bacillus cereus spores and Escherichia coli O157:H7 are reported. Both assays involve the use of dual (QSY-7 and Oregon Green 514-antibody)-labeled spores or vegetative bacteria, such that Oregon Green 514-labeled antibodies are quenched by proximal QSY-7 molecules that are covalently bound to the dual (Oregon Green 514 and QSY-7)-labeled cells. Upon introduction of unlabeled bacteria or spores, in the respective assays, an increase in fluorescence is observed in proportion to the numbers of unlabeled cells. This is due to migration of Oregon Green 514-labeled antibody from the dual-labeled cells to the unlabeled target cells as verified by fluorescence microscopy. Optimization of the QSY-7 surface density led to a B. cereus spore detection sensitivity of approximately 1.0 x 10(5) to 2.5 x 10(5) spores per milliliter and 3.5 x 10(5) cells per milliliter for E. coli using a conventional cuvette-based spectrofluorometer.

Bacillus cereus↗
PubMed2005

Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease.

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (K(i) = 4 microM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC(50) values ranging from 7.6 to 84.5 microM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

Binding Sites↗
PubMed2005

A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.

Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca(2+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca(2+) concentration ([Ca(2+)](i)) was raised. F2C had a Ca(2+) sensitivity with an apparent dissociation constant (K(d) for Ca(2+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca(2+)](i) was measured simultaneously. These properties of F2C are useful to be a Ca(2+) indicator.

Amino Acid Sequence↗
PubMed2006

Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.

cAMP-dependent protein kinase (PKA) mediates key cellular processes via compartmentalized activity, and the ability to track its activity in living cells should help increase our understanding of this precise regulation. Here, through systematic testing of new fluorescent proteins, we developed a new FRET-based A-kinase activity reporter (AKAR), AKAR3, with a dynamic range of 31-41%, twice that of predecessors. Visualization of PKA activity at plasma membrane, cytoplasm, nucleus, and mitochondria was achieved. Targeting AKAR3 to outer mitochondrial membrane revealed that basal PKA activity at mitochondria differs from that in the cytoplasm, indicating differential regulation of PKA activity at different subcellular locations.

Amino Acid Sequence↗
PubMed2004

Structural information on nanomolecular systems revealed by FRET.

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D-A distance distribution function rhoDA(r). We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers.

Algorithms↗
PubMed2005

Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus.

Genetically encoded sensors are becoming a powerful tool for investigating cellular signaling pathways and, potentially, signaling in vivo. Many sensors use changes in fluorescence resonance energy transfer (FRET) between donor and acceptor variants of GFP separated by a ligand binding domain sensitive to a particular signaling pathway. Accurate measurements require that sensors be insensitive to extraneous intracellular environmental factors. We have found that the responsiveness of the Ca(2+) sensor, cameleon YC6.1, varies linearly with the resting YFP/CFP emission ratio in the cell. However, cells expressing responsive or non-responsive sensor can easily be segregated by determining a resting YFP/CFP ratio cutoff for the sensor. This environmental sensitivity has been eliminated by replacing EYFP with Venus to produce a new cameleon we have designated VC6.1. Measurements show that VC6.1 has a greater dynamic range than YC6.1 and better environmental resistance. We also show that YC6.1 is inactivated by persistent activation of the IP(3) pathway following expression of constitutively active G(q), while VC6.1 is not. The stability of VC6.1 may make it well suited to studies utilizing mixed cell populations such as those encountered in vivo.

Animals↗
PubMed2005

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries.

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

Combinatorial Chemistry Techniques↗
PubMed2004

The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP.

Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 [Mol. Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group Ia, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg(2+) on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg(2+). Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4.

Amino Acid Sequence↗
PubMed2004

Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells.

Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2 beta, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2 beta, and MAN1 in living HeLa cells. Like endogenous BAF, GFP-BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP-BAF diffused rapidly; the halftimes for recovery in a 0.8 microm square area were 260 ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2 beta, and MAN1 were all relatively immobile, with recovery halftimes of about 1 min, for a 2 microm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP-BAF were distinctly regulated, with nuclear GFP-BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP-BAF binds directly to YFP-emerin at the inner nuclear membrane of living cells. We propose a "touch-and-go" model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP-BAF and GFP-emerin during telophase, when they colocalized at the 'core' region of telophase chromosomes at early stages of nuclear assembly.

Animals↗
PubMed2005

Rapid single tube genotyping of ACP1 by FRET based amplification and dual color melting curve analysis.

Erythrocyte acid phosphatase (ACP1), also named low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme involved in signal transduction pathways of tyrosine kinase receptor. The precise physiological role of ACP1 remains to be elucidated, however recent advancements suggest that it may play an important role in the control of cell proliferation. ACP1 is a highly polymorphic enzyme that has been investigated by case-control studies for decades. Initially based on protein electrophoresis, the phenotype of ACP1 is now detected by DNA-based techniques. Here, we report a new rapid single tube genotyping method for ACP1 by FRET based amplification and dual color melting curve analysis. This method does not require a post-procedure amplification process and allows unambiguous genotyping of 30 samples in less than 1 h.

Color↗
PubMed2005

A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes.

Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.

Animals↗
PubMed2005

Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.

Cell Line, Tumor↗
PubMed2005

A homogeneous FRET assay system for multiubiquitin chain assembly and disassembly.

Ubiquitin (Ub, 76aa) is a small highly conserved protein present universally in eukaryotic cells. Covalent attachment of (Ub)(n) to target proteins is a well-known posttranslational modification that has been implicated in a wide array of cellular processes including cell biogenesis. Ubiquitin polymerization by the Ub activation-conjugation-ligation cascade and the reverse disassembly process catalyzed by Ub isopeptidases largely regulate substrate protein targeting to the 26S proteasome. Ub chains of four or more subunits attached by K48 isopeptide linkages have been shown to be necessary for the 26S proteasome association and subsequent degradation of protein molecules. To better understand this protein degradation event, it is important to develop Ub polymerization and depolymerization assays that monitor every reaction step involved in Ub attachment to, or detachment from, substrate protein molecules. In this chapter, we describe homogeneous, easy-to-use, nonradioactive, complementary continuous fluorescence assays capable of monitoring the kinetics of Ub chain formation by E3 Ub ligases, and their hydrolysis by isopeptidases, which rely on mixing a 1:1 population of fluorophore-labeled Ub molecules containing a FRET pair. The proximity of fluorescein (donor) and tetramethylrhodamine (acceptor) in Ub polymers results in fluorescein quenching on ligase-induced Ub chain assembly. Conversely, a dramatic enhancement of fluorescein emission was observed on Ub chain disassembly because of isopeptidase activity. These assays thus provide a valuable tool for monitoring Ub ligase and isopeptidase activities using authentic Ub monomers and polymers as substrates. Screening of a large number of small molecule compound libraries in a high-throughput fashion is achievable, warranting further optimization of these assays.

Base Sequence↗
PubMed2000

A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin.

The molecular motor myosin is proposed to bind to actin and swing its light-chain binding region through a large angle to produce an approximately 10 nm step in motion coupled to changes in the nucleotide state at the active site. To date, however, direct dynamic measurements have largely failed to show changes of that magnitude. Here, we use a cysteine engineering approach to create a high resolution, FRET-based sensor that reports a large, approximately 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.

Adenosine Diphosphate↗
PubMed2003

Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy.

G protein-coupled potassium channels (GIRK/Kir3.x) are key determinants that translate inhibitory chemical neurotransmission into changes in cellular excitability. To understand the mechanism of channel activation by G proteins, it is necessary to define the structural rearrangements in the channel that result from interaction with Gbetagamma subunits. In this study we used a combination of fluorescence spectroscopy and through-the-objective total internal reflection microscopy to monitor the conformational rearrangements associated with the activation of GIRK channels in single intact cells. We detect activation-induced changes in FRET consistent with a rotation and expansion of the termini along the central axis of the channel. We propose that this rotation and expansion of the termini drives the channel to open by bending and possibly rotating the second transmembrane segment.

Animals↗