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At least 145 records · Page 8Linked to original sources
PubMed2005

In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft.

Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P(3)), and Ca(2+)/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca(2+) concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca(2+)-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins.

Binding Sites↗
PubMed2006

Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening.

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5+/-1.5% (mean+/-SEM) and 8.0+/-0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8+/-2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1+/-1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.

Cell Separation↗
PubMed2004

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye.

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.

DNA Primers↗
PubMed2004

Using live FRET imaging to reveal early protein-protein interactions during T cell activation.

The emerging challenge for proteomics in general and lymphocyte biology in particular is to understand protein-protein interactions in the dynamic context of the living cell. Particularly interesting are the molecular dynamics of the T cell receptor-CD3 complex and other immunoreceptors in immune synapses. Fluorescence (or Förster) resonance energy transfer (FRET) is one of the few techniques that are capable of giving dynamic information about the nanometer-range proximity between molecules, as opposed to simply the subcellular co-localization that is provided by fluorescence microscopy. Spectral changes in fluorescence intensity and down modulation of donor lifetime are the basis for rapidly developing approaches to real-time FRET imaging. With two-photon excitation, FRET can now be extended to in vivo imaging.

Animals↗
PubMed2006

Effect of electrostatic interactions on binding and retention of DNA oligomers to PNA liposomes assessed by FRET measurements.

A FRET-based method is used to observe the desorption of di-alkyl peptide nucleic acid amphiphiles (PNAA) from liposomes occurring on binding of complementary DNA oligomers. PNA liposomes were prepared containing fluorescein-labeled PNAA and rhodamine-labeled dipalmitoylphosphoethanolamine (DPPE). These liposomes showed efficient energy transfer from the fluorescein to rhodamine, with an average donor-to-acceptor distance of 5.91nm. In low-ionic-strength buffer (50mM Tris-HCl, pH 8.0), the FRET signal was maintained in the presence of a stoichiometric amount of 10- and 20-mers DNA complements, but the signal attenuated for 40-mer complements, indicating that DNA first binds the PNAA before the PNAA/DNA duplex desorbs from the lipid bilayer. The FRET signal was maintained in the presence of 10-, 20-, 40-, and 60-mer DNA in high ionic-strength buffer, showing that the driving force for the desorption is electrostatic repulsion between the bound DNA oligomer and the liposome surface. This conclusion is corroborated by comparison of the PNA/DNA binding energy, the energy of adsorption of the di-alkyl PNAA to the lipid bilayer, and a calculation of the DNA/lipid bilayer electrostatic repulsion using the linearized Poisson-Boltzmann equation.

Biosensing Techniques↗
PubMed2005

Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements.

The plant V-ATPase is a protein complex of 13 different VHA-subunits and functions as ATP driven motor that electrogenically translocates H+ into endomembrane compartments. The central rotor extends into the hexameric head that is fixed by peripheral stators to an eccentric membrane domain. The localization and orientation of VHA-subunits of the head and peripheral stalk region were investigated by in vivo fluorescence resonance energy transfer (FRET). To this end, VHA-E, VHA-G, VHA-H of the peripheral stalks as well as subunits VHA-A and VHA-B were C-terminally fused to cyan (CFP) and yellow fluorescent protein (YFP). Protoplasts transfected with FRET-pairs of CFP-donor and YFP-acceptor fluorophores fused to VHA-subunits were analysed for FRET by laser scanning microscopy. The result of the C-termini mapping allows to refine the arrangement and interaction of the subunits within the V-ATPase complex in vivo. Furthermore, expression of fused VHA-E and VHA-H stimulated acidification of protoplast vacuoles, while other constructs had no major effect on vacuolar pH tentatively indicating a regulatory role of these subunits in plants.

Amino Acid Sequence↗
PubMed2006

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

Cells, Cultured↗
PubMed2004

FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery.

The intracellular degradation of single-stranded, double-labeled oligonucleotides (ONs) was studied by following the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the rhodamine green and Cy5 fluorophores attached to respectively the 3' and 5' end of the ONs. The green and red fluorescence intensities upon rhodamine green excitation were monitored using the ultra-sensitive detectors of a dual-color Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements showed to give accurate information on the integrity of the ONs, without the need for additional auto- or cross-correlation analysis of the registered fluorescence intensity fluctuations. Intracellular measurements revealed that most of the 40mer phosphodiester ONs were degraded before they entered the nucleus. For the 20mer phosphodiester ONs, this degradation occurred more slowly, and both intact and degraded ONs entered the nucleus. For the 20mer phosphorothioate ONs, no intracellular degradation was observed during the measured time period. The sensitive detection of the intracellular fluorescence by the FCS setup will be particularly useful in situations where the expected fluorescence is too low to be detected by FRET-imaging as may occur after intracellular delivery of ONs by cationic carriers.

Animals↗
PubMed2006

Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.

We demonstrate a highly sensitive method to characterize the structural composition and intracellular fate of polymeric DNA nanocomplexes, formed by condensing plasmid DNA with cationic polymers through electrostatic interactions. Rational design of more efficient polymeric gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the non-viral gene transfer process. To characterize the composition and binding dynamics of nanocomplexes, plasmid and its polymer carrier within nanocomplexes were labeled with quantum dots (QDs) and fluorescent organic dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled precise detection of discrete changes in nanocomplex state at the single-particle level, against various intracellular microenvironments. The distribution and unpacking of individual nanocomplexes within cells could thus be unambiguously followed by fluorescence microscopy. QD-FRET is a highly sensitive and quantitative method to determine the composition and dynamic stability of nanocomplexes during intracellular transport, where barriers to gene delivery may be identified to facilitate gene carrier optimization.

Cell Line, Tumor↗
PubMed2005

Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein.

The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.

Anilino Naphthalenesulfonates↗
PubMed2006

Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations.

Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li(+), Na(+), K(+), NEt(4)(+) and Mg(2+)) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5'- and 3'- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg(2+) cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na(+) and K(+) ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.

Anisotropy↗
PubMed2002

Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.

Animals↗
PubMed2003

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Animals↗
PubMed2003

FRET-based in vivo screening for protein folding and increased protein stability.

Fluorescence resonance energy transfer (FRET) was used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the cytoplasm of Escherichia coli. The system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue-fluorescent protein (BFP) to the N terminus of protein X. Efficient FRET from BFP to GFP in the ternary fusion protein is observed in vivo only when protein X is folded and brings BFP and GFP into close proximity, while FRET is lost when BFP and GFP are far apart due to unfolding or intracellular degradation of protein X. The screening system was validated by identification of antibody V(L) intradomains with increased thermodynamic stabilities from expression libraries after random mutagenesis, bacterial cell sorting, and colony screening.

Amino Acid Sequence↗
PubMed2003

Can Förster resonance energy transfer measurements uniquely position troponin residues on the actin filament? A case study in multiple-acceptor FRET.

Straightforward interpretation of Förster resonance energy transfer (FRET) data in terms of the distance from donor-labeled troponon-tropomyosin (TnTm) to acceptor-labeled actin is complicated by the potential for energy transfer to acceptors on neighboring actin monomers (cross-transfer). Calculations indicate that cross-transfer can account for a substantial percentage of the total transfer efficiency. In some cases, this renders isolated FRET data uninterpretable. To overcome these limitations, we have developed an analysis method that incorporates cross-transfer and can, in principle, define the most probable (in the "least-squares" sense) position of a TnTm residue on the actin filament. The technique analyzes data from four or more FRET experiments using acceptors attached to different residues on actin. We have used this method to specify the coordinates of skeletal troponin I (sTnI) residue 133 relative to the actin filament under Mg(2+) and Ca(2+)-saturating conditions. Ca(2+)-activation causes the C terminus of the regulatory domain of TnI to move away from the actin surface by 6.3A, laterally along the actin surface toward actin subdomain 3 by 22.0A, and azimuthally toward the actin inner domain by 13.2A. This information is used to construct a low-resolution structural model of thin filament activation.

Actins↗
PubMed2001

Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.

Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+), acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca(2+)-insensitive mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca(2+)-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.

Calcium↗
PubMed2001

Recent advances in FRET: distance determination in protein-DNA complexes.

Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A. Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling. Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology.

Bacterial Proteins↗
PubMed2006

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration.

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.

Binding Sites↗