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At least 235 records · Page 13Linked to original sources
PubMed1998

Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.

A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.

Binding Sites↗
PubMed1999

A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology.

A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.

Chromium↗
PubMed2003

Structural study of melanocortin peptides by fluorescence spectroscopy: identification of beta-(2-naphthyl)-D-alanine as a fluorescent probe.

Several cyclic disulfide alpha-melanocyte stimulating hormone (alpha-MSH) analogues containing the aromatic fluorescent amino acid beta-(2-naphthyl)-D-alanine (D-Nal) have high affinity and selectivity for the melanocortin (MC)-4 receptor. Considering the possible relevant role played by the lipid phase in the peptide-receptor interaction, the structures of two cyclic alpha-MSH analogues, containing both Trp and D-Nal fluorophores, were investigated by steady-state and time-resolved fluorescence spectroscopy, in aqueous solution and in the presence of dimyristoyl phosphatidylglycerol (DMPG) vesicles, and compared with that of the natural peptide. The amino acid D-Nal gives a unique de-excitation fluorescence profile, with an excited state lifetime much longer than those of Trp, allowing good distinction between the two fluorophores. The cyclic analogues' aqueous structures seem to be adequate for membrane penetration, as Trp fluorescence indicates that, in both aqueous and lipid media, the Trp environment in the cyclic peptides is similar to that of alpha-MSH when incorporated in lipid bilayers. Trp, in the cyclic analogues, seems to penetrate deeper in the bilayer than in the native peptide. The amino acid D-Nal was also found to penetrate deep into the lipid bilayer, having its excited-state lifetime drastically changed from aqueous solution to lipid medium. The present work shows that D-Nal may serve as a fluorescent probe for studies of MC peptides and suggests that the high affinity and selectivity of the cyclic peptides to the MC4 membrane receptor could be related to their deeper penetration into the bilayer core.

Alanine↗
PubMed2003

Synthesis of a high-affinity fluorescent PPARgamma ligand for high-throughput fluorescence polarization assays.

Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investigation as molecular targets for the treatment of numerous diseases including Alzheimer's, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARgamma subtype complexed with the potent small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein. This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor affinity and maximal effects on fluorescence polarization properties. The recombinant PPARgamma ligand binding domain protein bound tightly and specifically to this probe with Kd=61+/-14 nM as determined by FP measurements. Competition binding assays with known PPARgamma ligands provided Ki values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. This fluorescent PPARgamma probe enables high-throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARgamma ligands, and this rational ligand design approach may be applied to other therapeutically important members of the nuclear hormone receptor superfamily.

Animals↗
PubMed2000

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C.

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.

Chlorophyll↗
PubMed2004

Acidity effects on the fluorescence properties and adsorptive behavior of rhodamine 6G molecules at the air-water interface studied with confocal fluorescence microscopy.

The effects of acidity on fluorescence originated from rhodamine 6G (R6G) molecules adsorbed at the air-water interface of extremely low-concentration aqueous solutions have been studied with confocal fluorescence microscopy. Similarities and differences in the observed acidity effects between R6G molecules at the interface and those in the bulk solution have been discussed. With increasing the subphase-pH from 1 to 6, height and frequency of photon bursts as well as intensity of the interface-originated fluorescence change in two steps, while bulk fluorescence changes in one step and a little change in the number of adsorbed R6G molecules is verified with surface tension measurements. The results suggest that there is an interface-specific equilibrium among the chemical forms of R6G molecules. Chemical forms contributing to the interface-originated fluorescence above pH 5 are discussed.

Adsorption↗
PubMed2000

Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.

We describe herein fluorescent indicators for cyclic GMP (cGMP) in single living cells. cGMP-dependent protein kinase Ialpha (PKG Ialpha), a receptor for cGMP, was fused with blue- and red-shifted green fluorescent proteins (GFPs) to its N- and C-termini, respectively. Using PKG lalpha delta1-47, in which the dimerization domain was deleted, fluorescence resonance energy transfer between the GFPs was found to increase upon cGMP-induced conformational change in PKG Ialpha delta1-47. We demonstrated that thus-developed fluorescent indicators reversibly responded to cGMP that was produced in nitric oxide-stimulated HEK293 cells. The present genetically encoded fluorescent indicators open a way not only for understanding the dynamics of cGMP signaling in single cells and organisms but also for discovering pharmaceuticals such as isoform-specific inhibitors for phosphodiesterases.

Animals↗
PubMed1985

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).

Bacterial Proteins↗
PubMed2005

Reactant concentrations from fluorescence correlation spectroscopy with tailored fluorescent probes. An example of local calibration-free pH measurement.

The present account is concerned with the measurement of local reactant concentrations by observing specific fluorescent probes in fluorescence correlation spectroscopy (FCS). The Theoretical Analysis section revisits the photophysical, thermodynamic, and kinetic information that is contained in the corresponding FCS correlation curves. In particular, we examine the conditions under which FCS is revealed as a superior tool to measure concentrations of reactive species. Careful molecular engineering of the specific fluorescent probes that simultaneously integrates photophysical, thermodynamic, and kinetic constraints will be required to benefit most from FCS. We illustrate the FCS titration approach with a series of fluorescent probes that we tailored to measure pH at around 4-6 by FCS after two-photon excitation. We show that an optimal design allows one to access pH without any preliminary calibrations such as the determination of the protonation constant or the photophysical properties of the fluorescent probe.

Fluorescent Dyes↗
PubMed2006

Probing the decay coordinate of the green fluorescent protein: arrest of cis-trans isomerization by the protein significantly narrows the fluorescence spectra.

The fluorescence spectra of the wild-type green fluorescence protein (wt-GFP) and the anionic form of p-hydroxybenzylidenedimethylimidazolone (p-HBDI), which models the protein chromophore, were obtained in the 80-300 K temperature range in glycerol/water solvent. The protein spectra have pronounced and well-resolved vibronic structure, at least at lower temperatures. In contrast, the chromophore spectra are very broad and structureless even at the lowest temperatures. Analysis of the spectra shows that the experimentally observed red-shift of the protein spectrum upon heating is apparently caused by quadratic vibronic coupling of the torsional deformation (TD) of the phenyl single bond of the chromophore to the electronic transition. The broad spectra of the chromophore manifest the contribution of different conformations in the glycerol/water solvent. In particular, the lowest-temperature spectrum reflects the distribution over the same TD coordinate in the excited electronic state, which essentially contributes to the asymmetry of the spectrum. Upon heating, motion along this coordinate leads to a configuration from which the radiationless transition takes place. This narrows the distribution along the TD coordinate, causing a more symmetric fluorescence spectrum. We were able to reconstruct the broad, structureless fluorescence spectra of p-HBDI in glycerol/water solutions at various temperatures by convoluting the original wt-GFP spectra with the function describing the distribution of the transition energies of the p-HBDI chromophore. Thus, both the fluorescence broadening and increase in radiationless transition upon removal of the protein chromophore to bulk solvent are consistent with decay by a barrierless TD of the phenyl single bond.

Benzylidene Compounds↗
PubMed1998

Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants.

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

Arabidopsis↗
PubMed2006

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.

Animals↗
PubMed2002

Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell.

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity-based FRET microscopy is limited by spectral bleed-through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed-through is not an issue in FRET-FLIM imaging. In this paper we describe the development of a nanosecond FRET-FLIM microscopy instrumentation to acquire the time-resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one- and two-component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.

CCAAT-Enhancer-Binding Protein-alpha↗
PubMed2001

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate.

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3' region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20-25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Animals↗
PubMed1999

Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy.

Fluorescence correlation microscopy (FCM) was applied to characterize fusion proteins of the green fluorescent protein (GFP) on the cellular as well as molecular level within seconds in an integrated instrument. FCM combines the inherent sensitivity and high spatial resolution of fluorescence correlation spectroscopy with fluorescence imaging and micropositioning, thereby providing a spectrum of molecular information in the cellular context. Signatures of characteristic parameters derived from the autocorrelation functions served to distinguish a GFP fusion protein of the epidermal growth factor receptor from GFP fluorescence in the endoplasmic reticulum and cytoplasm. Diffusion constants measured for free transiently expressed GFP reproduced values reported previously with other techniques. The accessible concentration range extends from millions to only a few thousand molecules per cell, with single molecule detectability in the femtoliter detection volume. The detailed molecular characterization offered by FCM is fully compatible with automation in sample identification and detection, offering new possibilities for highly integrated high-throughput screening.

Animals↗
PubMed1998

Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.

Fluorescence↗
PubMed1976

Fluorescent probes for antibody active sites. I. Production of antibodies specific to the N-methyl-2-anilinonaphthalene-6-sulfonyl group in rabbits and some fluorescent properties of the hapten bound to the antibodies.

1. Rabbits were immunized with N-methyl-2-anilinonaphthalene-6-sulfonyl (MANS) bovine serum albumin (MANS-BSA) prepared by the reaction of bovine serum albumin with N-methyl-2-anilinonaphthalene-6-sulfonyl chloride to obtain IgG fraction containing anti-MANS antibodies. 2. Both N-methyl-2-anilinonaphthalene-6-sulfonate (MANSate) and N-methyl-2-anilinonaphthalene-6-sulfonamide (MANSamide), which are virtually non-fluorescent in aqueous solution, became strongly fluorescent, with a blue shift of about 100 nm, when they were specifically bound to the antibodies against the hapten group contained in the IgG fractions. 3. IgG fractions were obtained from five immunized rabbits at intervals after the primary immunization. The fluorescence characteristics of the hepten were carefully examined with preparations of IgG fraction differing in source and time of bleeding. One of the rabbits showed a very interesting immune response. The rabbit produced two groups of anti-MANS antibodies which were significantly different in their effect on the fluorescence properties of the hapten group. One of the groups was exclusively produced at the early stage in the immune response. The other group appeared at the late stage. It is suggested that the two groups differ as regards the structure of the hepten combining sites. 4. The above findings indicate that the MANS group can be successfully used as a hapten with useful properties as a fluorescent probe.

Anilino Naphthalenesulfonates↗
PubMed1983

Fluorescence spectroscopy, fluorescence polarization, and circular dichroism in studies on pH-dependent changes in the alpha-fetoprotein molecule.

Conformational transitions of human alpha-fetoprotein (AFP) in the pH range from 2 to 12 were studied by fluorescence spectroscopy, fluorescence polarization, and circular dichroism in order to compare the molecular architecture of AFP with that of human serum albumin (HSA). In a previous paper we have found that AFP has a remarkably hydrophilic exposed molecular surface at neutral pH and possesses extensive hydrophobic binding sites located in crevices. Conformational changes of AFP occur in the acid and alkaline pH regions; extensive, hydrophobic areas of AFP are exposed by both acid and alkaline transitions. Similar results were obtained using optical methods. Both fluorescence and CD measurements disclosed conformational transitions of AFP induced by acidic or alkaline pH. Changes of the fluorescence intensity of AFP and HSA were compared with the model substances, that is, with lysozyme and Gly-Trp dipeptide. Again AFP and HSA show similar features, especially at the neutral pH and during alkaline transition. In the acid pH region, decrease of the AFP emission fluorescence intensity was greater than that of HSA and evidence for some irreversible conformational changes of AFP was obtained. CD spectra of both proteins also show a very similar pattern. The changes of molar ellipticity with pH for HSA are very much like those found for AFP. We have estimated high alpha-helix content--67% at pH 7.6 and 47% at pH 2.11. These figures are very close to those given for bovine albumin and rat alpha-fetoprotein. These findings provide additional support for our former findings that the molecular architecture of human alpha-fetoprotein has features similar to those of human serum albumin.

Circular Dichroism↗