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At least 217 records · Page 12Linked to original sources
PubMed1998

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

Animals↗
PubMed2002

Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag.

Gene therapy employing the suicide gene/prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) is effective in killing malignant tumor cells. Labeling of the HSV-TK enzyme with fluorescent proteins makes possible the non-invasive imaging of transduction efficiency, enzyme localization and activity in cell culture and in animal models of human cancers. Here we report the expression of HSV-TK tagged with different fluorescent proteins (EGFP, DSRed1, DsRed2, dsdrFP616) and show that intracellular localization of the fusion products depends on the nature of the fluorescent tag despite the presence of several nuclear targeting signals within the enzyme itself. Coexpression of red fluorescent HSV-TK fusion proteins with TK-EGFP or untagged HSV-TK allowed these proteins to enter the nucleus by inhibiting formation of red fluorescent protein oligomers. As enzyme localization may influence HSV-TK activity, this observation is of potential importance to gene therapy studies.

3T3 Cells↗
PubMed1999

Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane.

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Bacteriophage T4↗
PubMed2006

Ultrafast excited-state dynamics of DNA fluorescent intercalators: new insight into the fluorescence enhancement mechanism.

The excited-state dynamics of the DNA bisintercalator YOYO-1 and of two derivatives has been investigated using ultrafast fluorescence up-conversion and time-correlated single photon counting. The free dyes in water exist in two forms: nonaggregated dyes and intramolecular H-type aggregates, the latter form being only very weakly fluorescent because of excitonic interaction. The excited-state dynamics of the nonaggregated dyes is dominated by a nonradiative decay with a time constant of the order of 5 ps associated with large amplitude motion around the monomethine bridge of the cyanine chromophores. The strong fluorescence enhancement observed upon binding of the dyes to DNA is due to both the inhibition of this nonradiative deactivation of the nonaggregated dyes and the dissociation of the aggregates and thus to the disruption of the excitonic interaction. However, the interaction between the two chromophoric moieties in DNA is sufficient to enable ultrafast hopping of the excitation energy as revealed by the decay of the fluorescence anisotropy. Finally, these dyes act as solvation probes since a dynamic fluorescence Stokes shift was observed both in bulk water and in DNA. Very similar time scales were found in bulk water and in DNA.

DNA↗
PubMed2001

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection.

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.

Animals↗
PubMed1999

Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.

A Kinase Anchor Proteins↗
PubMed2006

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions.

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.

Animals↗
PubMed2002

Monitoring of chemical and enzymatic hydrolysis of water-soluble proteins using flow-injection analysis with fluorescence detection and an aqueous eluant containing 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe.

The exposed hydrophobicity of proteins, which is due to the hydrophobic regions located on their surfaces, enhances the fluorescence intensity of the probe 2-p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) by the formation of a complex. During the hydrolysis of a protein, the average exposed hydrophobicity of the substrate continuously changes with incubation time, and these changes are immediately reflected by a corresponding change in the fluorescence intensity of the 2,6-TNS/substrate complex. Therefore, 2,6-TNS seems to be a good probe to monitor the course of the depolymerization processes of proteins. In this work, bovine serum albumin and alpha-casein have been hydrolyzed both chemically and enzymatically, and the course of the reactions is monitored by using flow-injection analysis (FIA) with fluorescence detection and a buffered aqueous eluant containing 2,6-TNS as the fluorescent probe. Results indicate that the time evolution of the fluorescence intensity of the 2,6-TNS/substrate complex can be correlated with the initial concentration of the parent protein, in mass per unit volume, the hydrolytic activity added, and the time evolution of the mean chain length of the substrate. In addition, because the time elapsed between injection of the sample into the FIA system and measurement of the corresponding fluorescence intensity is only a few seconds, this methodology could be a useful tool for on-line monitoring of processes for the production of protein hydrolysates.

Biosensing Techniques↗
PubMed2001

Microenviromental investigation of polymer-bound fluorescent chelator by fluorescence microscopy and optical spectroscopy.

8-Hydroxyquinoline-5-sulfonic acid (HQS) was immobilized onto a strong-base anion-exchange resin AG MP-1 for the purpose of microenvironment investigation, resin characterization, and possibly sensing cadmium. The maximum loading of HQS was found to be 0.9340 mmol/g of AG MP-1. A plateau for Cd complex capacity was already obtained for 0.5500 mmol of HQS/g of AG MP-1. A minicolumn experiment showed an influence of influent Cd concentration on column capacity. IR and Raman spectra proved an electrostatic mode for HQS immobilization and Cd complex formation. UV spectroscopy showed significant differences between solution and solid state for both HQS and Cd complex. A fluorescence microscopy technique was used for fluorescence spectral measurement, microdistribution imaging, and study of photobleaching of HQS and the HQS-Cd complex in the resin phase. The fluorescence of immobilized HQS was found to be red-shifted with regard to the solid-state HQS. The microdistribution of uncomplexed and Cd-complexed AG MP-1-HQS was directly visualized by fluorescence imaging, showing a nonuniform distribution. Cadmium complexation modifies the fluorescence emission of uncomplexed AG MP-1-HQS, exhibiting an increased and red-shifted emission. Significant photobleaching of the fluorescence from the Cd complex was recorded, indicating the occurrence of photochemical reactions within the microenvironment of the resin phase.

Journal Article↗
PubMed2001

On the relationship between the non-photochemical quenching of the chlorophyll fluorescence and the Photosystem II light harvesting efficiency. A repetitive flash fluorescence induction study.

Plants respond to excess light by a photoprotective reduction of the light harvesting efficiency. The notion that the non-photochemical quenching of chlorophyll fluorescence can be reliably used as an indicator of the photoprotection is put to a test here. The technique of the repetitive flash fluorescence induction is employed to measure in parallel the non-photochemical quenching of the maximum fluorescence and the functional cross-section (sigma(PS II)) which is a product of the photosystem II optical cross-section a(PS II) and of its photochemical yield Phi(PS II) (sigma (PS II) = a(PS II) Phi(PS II)). The quenching is measured for both, the maximum fluorescence found in a single-turnover flash (F(M) (ST)) and in a multiple turnover light pulse (F(M) (MT)). The experiment with the diatom Phaeodactylum tricornutum confirmed that, in line with the prevalent model, the PS II functional cross-section sigma (PS II) is reduced in high light and restored in the dark with kinetics and amplitude that are closely matching the changes of the F(M) (ST) and F(M) (MT) quenching. In contrast, a poor correlation between the light-induced changes in the PS II functional cross-section sigma (PS II) and the quenching of the multiple-turnover F(M) (MT) fluorescence was found in the green alga Scenedesmus quadricauda. The non-photochemical quenching in Scenedesmus quadricauda was further investigated using series of single-turnover flashes given with different frequencies. Several mechanisms that modulate the fluorescence emission in parallel to the Q(A) redox state and to the membrane energization were resolved and classified in relation to the light harvesting capacity of Photosystem II.

Journal Article↗
PubMed2003

Effect of heat on phycoerythrin fluorescence: Influence of thermal exposure on the fluorescence emission of R-phycoerythrin.

The goal of this work was to measure and model the effect of thermal exposure on the fluorescence emission of R-phycoerythrin (R-PE). The long-term objective of our work is to assess the feasibility of encapsulating R-PE for use as the critical component of a time-temperature integrator (TTI) for ascertaining the degree of inactivation of food pathogens such as Salmonella. In this article we present a study to measure and model the thermally induced fluorescence emission decay of R-PE in several isothermal experiments. We used the isothermal data to determine the kinetic parameters, based on a general n(th) order reaction, and evaluated the utility of the resulting model by using it to predict R-PE fluorescence emission decay for several nonisothermal experiments based on published USDA safe harbor guidelines for cooked beef products. The transient experiments were conducted over the same temperature range used in the isothermal study. Very good agreement was obtained between theory and experiment at temperatures of 62.8 degrees C and above, although the model slightly underpredicted the extent of fluorescence emission decay at 60 degrees C. Our results indicate that R-PE fluorescence emission decay kinetics is well behaved and that the protein is a strong candidate for use as a time-temperature integrator.

Biosensing Techniques↗
PubMed1995

Combined light and fluorescent microscopical imaging of nucleolar organizer regions and cellular rRNA as detected by fluorescent in situ hybridization.

A method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag-NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG-63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag-NOR within individual cells, brightfield images were digitized, and the total Ag-NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag-NOR procedure did not significantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag-NOR. With this method, both Ag-NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag-NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag-NOR indices with cellular proliferation.

Fibrosarcoma↗
PubMed1999

One- and two-photon-induced fluorescence from recombinant green fluorescent protein.

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.

Anisotropy↗
PubMed1997

A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures.

Green fluorescent protein (GFP) from Aequorea victoria is widely used as a marker of gene expression and protein localization in living cells from prokaryotes to eukaryotes. However, the total fluorescent signal from wild-type GFP is very weak when expressed in cells cultured at 37 degrees C compared to 30 degrees C or below. This characteristic makes GFP poorly suited to use as a marker in mammalian cells. Here we describe a new variant of GFP which carries a substitution of Ser147 to Pro (S147P GFP) and which emits a stronger fluorescent signal than the wild-type GFP at high temperature. When S147P is combined with the Ser65 to Thr mutation (S65T GFP), the resulting double mutant emits fluorescence which is several-fold stronger than GFP with a single S65T modification in both bacterial or mammalian cells. This S147P mutation should be useful for constructing new GFP variants which stably emit strong fluorescence at a wide range of culturing temperatures.

Fluorescence↗
PubMed1990

New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.

16 fluorochromes were examined for their ability to label viable lymphocytes in vitro and yield fluorescence detectable by fluorescence microscopy and flow cytometry. Of these fluorochromes, four intracellular dyes were found to be suitable for in vivo migration studies. They were H33342, the well known DNA-binding dye which excites and emits in the UV range, and three fluorescein based cytoplasmic dyes, namely BCECF-AM, Calcein-AM and CFSE which excite and emit in the visible range. Lymphocytes labelled with H33342, BCECF-AM and Calcein-AM were suitable for short term in vivo migration experiments with detection by flow cytometry 2-3 days post injection. In contrast lymphocytes labelled with CFSE, a fluorochrome which can covalently couple with intracellular macromolecules, were detected by flow cytometry up to 8 weeks post injection and thus this fluorochrome is ideal for long term migration experiments. Due to marked differences in fluorescence profiles, BCECF-AM and Calcein-AM could be used for short term double labelling experiments using the flow cytometer in which entry of injected lymphocytes into lymphoid organs was quantified. Similarly, in vivo localization of lymphocyte subpopulations could be examined by fluorescence microscopy utilizing differences in fluorescence excitation and emission spectra of lymphocytes labelled with H33342 and one of the fluorescein based dyes.

Animals↗
PubMed2005

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing.

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.

Acridines↗
PubMed2006

Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy.

The spatiotemporal localization of neuronal signaling is important for triggering neuronal responses in specific locations at precise times. Fluorescence resonance energy transfer imaging enables measurement of spatiotemporal dynamics of signaling activity in live neurons. Although the usefulness of fluorescence resonance energy transfer is well recognized, there are many difficulties in applying it, particularly when imaging in neuronal micro-compartments in light-scattering brain tissue. Fluorescence resonance energy transfer has been imaged using several techniques including intensity-based methods, fluorescence lifetime imaging and fluorescence anisotropy imaging. These methods have different advantages and disadvantages, and thus are suitable in different applications.

Animals↗