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PubMed2006

Excimer fluorescence as a tool for monitoring protein domain dynamics applied to actin conformation changes based on circulary polarized fluorescence spectroscopy.

Fluorescence-detected circular dichroism (FDCD) was introduced into the study of protein conformation changes. Actin was used as a model protein which undergoes dynamic conformation changes as it polymerizes. Actin labeled with N-(1-pyrene)iodoacetamide (PIA) showed monomer fluorescence peak at 386 and 410 nm, and excimer fluorescence peak at around 480 nm. Excimer was formed by PIA-dimers labeled to different sites of amino acid residues. New information concerned with actin structural changes were monitored by fluorescence emission spectra excited with left- and right-circulary polarized light at 355 nm. FDCD intensities were shown as the difference in the fluorescence emission DeltaF, where DeltaF=(F (L)-F (R))/(F (L)+F (R)) denoting F (L) and F (R) as emissions obtained by excitation with left- and right-circulary polarized light. When solvent conditions of PIA-actin were changed by addition of NaCl, TFE, or ATP, DeltaF showed sensitive responses to these compounds. From the analysis of DeltaF (M) and DeltaF (E) which represent the peaks of DeltaF at the monomer- and excimer-emission band, the information concerned with the actin intrastructural changes were obtained. This method based on monitoring the excimer fluorescence with FDCD could be used for other proteins to extract finer structural changes that cannot be detected by the normal fluorescence spectroscopy.

Actins↗

PubMed1986

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids.

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.

Estriol↗

PubMed2004

Using capillary electrophoresis with laser-induced fluorescence to study the interaction of green fluorescent protein-labeled calmodulin with Ca2+- and calmodulin-binding protein.

A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni(2+)-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2 x 10(-10) M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca(2+) was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K(d)) between GFP-CaM and Ca(2+) was determined to be 1.2 x 10(-5), which is in good agreement with the literature values of untagged CaM (10(-6) to 10(-5)M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.

Base Sequence↗

PubMed2004

Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy.

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.

Kinetics↗

PubMed2005

Scanning fluorescence correlation spectroscopy: a tool for probing microsecond dynamics of surface-bound fluorescent species.

In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16 and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules.

Fluorescent Dyes↗

PubMed1997

Dynamic fluorescence spectroscopy on single tryptophan mutants of EII(mtl) in detergent micelles. Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay.

The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme [Swaving Dijkstra et al. (1996) Biochemistry 35, 6628-6634]. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EII(mtl), respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns was resolved for tryptophan 30, which probably represents a rotational mode of the micelle-embedded C-domain of EII(mtl) or a portion thereof. Upon phosphorylation, this rotational correlation time increases to 50 ns, probably reflecting a changed spatial orientation of W30 with respect to the C domain. Although kinetic experiments have shown that none of the tryptophans is essential for the catalytic activity of EII(mtl), it is significant that the residues most sensitive to mannitol binding, W30 and W42, are both located in the first membrane-spanning alpha-helix, a portion of which is highly conserved among mannitol-specific EII's of different bacteria.

Detergents↗

PubMed2004

Intramolecular fluorescence quenching of crowned 7-aminocoumarins as potential fluorescent chemosensors.

The effects of the nature of solvent, temperature and complex formation with alkali and alkaline-earth metal cations, as well as protonation, on the efficiency and the kinetics of fluorescence of 3-azacrowned 7-diethylaminocoumarins have been studied. For the crown-ethers under investigation, the ratio of a dipole moment to the radius of Onsager cavity delta micro/rho is a constant value, and a macrocycle does not affect delta micro, and rho. The fluorescence of coumarin 1 in acetonitrile is quenched by an electron donor, triethylamine, with the Stern-Volmer constant being equal to (0.474+/-0.009) M(-1). The decrease in coumarin 1 fluorescence quantum yield upon the introduction of N-alkylazacrown moiety into position 3 is caused by an intramolecular photoinduced electron transfer from the nitrogen atom of macroheterocycle to the coumarin moiety, where the excitation is localized. The fluorescence quenching has an activation energy 2.32+/-0.05 kcal/mol in various hydrocarbons, and does not depend on the solvent viscosity. The fluorescence kinetics of free crowned coumarins in methanol is not monoexponential because of the existence of macrocycle conformers, or because of the hydrogen bond complex formation between the solvent and the nitrogen atom of macrocycle, in which the efficiency of intramolecular electron transfer is different. Upon complex formation with alkali and alkaline-earth metal cations and upon protonation, the fluorescence quantum yield increases and fluorescence decay becomes monoexponential.

Coumarins↗

PubMed2002

Fluorescence polarization of green fluorescence protein.

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60 degrees to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

Animals↗

PubMed2000

Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins.

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

Bacterial Proteins↗

PubMedDate not supplied

Temperature dependent fluorescence of A2-E, the main fluorescent lipofuscin component in the RPE.

PURPOSE: A2-E is the dominant fluorophore of lipofuscin in the retinal pigment epithelium. In an in-vitro setup, we determined the temperature-dependent changes of the A2-E fluorescence with the aim of also assessing the potential value of such measurements for determining retinal temperature by autofluorescence measurements during laser treatment. METHODS: A2-E was biosynthesized and diluted in Dimethyl Sulfoxide (DMSO) to 1 microM. Fluorescence measurements were performed with a photospectrometer under various temperatures ranging from 20 degrees C to 75 degrees C. Autofluorescence was excited at 467 nm, and emission was detected around 632 nm. RESULTS: A2-E fluorescence intensity showed a linear decrease concomitant with temperature increment. At 75 degrees C, the fluorescence intensity decreased by 43% compared to at 20 degrees C. Fluorescence intensity was completely reversible dependent on the temperature, which cannot be explained by thermal A2-E alteration. CONCLUSIONS: If the A2-E temperature-dependent fluorescence in-vitro is transferable to human fundus auto-fluorescence, then it may be possible to apply an autofluorescence-based online detection device for noninvasive determination of fundus temperature during in vivo laser treatment. This is of clinical relevance, especially for the application of photodynamic therapy (PDT) and transpupillary thermotherpy (TTT).

Fluorescence↗

PubMed2005

Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements.

Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.

17-alpha-Hydroxyprogesterone↗

PubMed2005

Two-point separation in far-field super-resolution fluorescence microscopy based on two-color fluorescence dip spectroscopy, Part I: Experimental evaluation.

The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.

Fluorescent Dyes↗

PubMed2005

[Studying on the relationship between the fluorescence occurring time and fluorescence summit wavelength].

The authors have detected the fluorescence characteristic of aromatic pollution material-anthracene by OMA and obtained its 3-D(time-wavelength-fluorescence intensity) fluorescence diagrams, three summit wavelengths's fluorescence time spectra, equal high spectra. By analyzing these spectra properties, the authors find that there is a clear order with the occurring time among the three fluorescence summit wavelengths (399 nm, 423 nm, 445 nm). Long fluorescence wave occurs earlier than short fluorescence wave. This solution is also analyzed and explained by atomic energy levels theory.

Algorithms↗

PubMed2000

Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy.

We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-galactosidase fusion protein in living cells with fluorescence correlation spectroscopy. Our fluorescence correlation spectroscopy device, in connection with a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuations in cell lines expressing EGFP are caused by molecular diffusion as well as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluorescent states that have to be taken into account when evaluating the fluorescence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluorescence correlation spectroscopy data can either be evaluated as a two component model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueous solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alternatively, we can fit the data to an anomalous diffusion model where the time dependence of the diffusion serves as a measure for the degree of obstruction, which is large especially in nuclei. Possible mechanisms for this long tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with recent numerical models of the chromosome territory structure in the cell nucleus.

Animals↗

PubMed2005

Improved routine bio-medical and bio-analytical online fluorescence measurements using fluorescence lifetime resolution.

Fluorescence techniques are widely used as sensitive detection methods in bio-analytics. The use of the bio-physical parameter fluorescence lifetime additional to the spectral characteristics of fluorescence has the potential to improve fluorescence-related detection methods in terms of selectivity in signal recognition, robustness against disturbing influences, and the accessibility of novel bio-chemical process parameters. This article describes the technical set up of a time-resolving instrument with either a fixed time-gated detection principle for improved evaluation of tissue metabolism by an online monitoring of the tissue autofluorescence or a direct fluorescence lifetime detection principle for lifetime-based fluorescent assays.

Animals↗

PubMed2004

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy.

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.

Animals↗

PubMed1979

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis.

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.

Animals↗

PubMed1976

Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins.

1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).

Binding Sites↗