[Show abstract][Hide abstract] ABSTRACT: The effect of violaxanthin and zeaxanthin, two main carotenoids of the xanthophyll cycle, on molecular organization of LHCII, the principal photosynthetic antenna complex of plants, was studied in a model system based on lipid-protein membranes, by means of analysis of 77 K chlorophyll a fluorescence and "native" electrophoresis. Violaxanthin was found to promote trimeric organization of LHCII, contrary to zeaxanthin which was found to destabilize trimeric structures. Moreover, violaxanthin was found to induce decomposition of oligomeric LHCII structures formed in the lipid phase and characterized by the fluorescence emission band at 715 nm. Both pigments promoted formation of two-component supramolecular structures of LHCII and xanthophylls. The violaxanthin-stabilized structures were composed mostly of LHCII trimers while, the zeaxanthin-stabilized supramolecular structures of LHCII showed more complex organization which depended periodically on the xanthophyll content. The effect of the xanthophyll cycle pigments on molecular organization of LHCII was analyzed based on the results of molecular modeling and discussed in terms of a physiological meaning of this mechanism. Supramolecular structures of LHCII stabilized by violaxanthin, prevent uncontrolled oligomerization of LHCII, potentially leading to excitation quenching, therefore can be considered as structures protecting the photosynthetic apparatus against energy loses at low light intensities.
Full-text · Article · Jan 2016 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: High antifungal activity is reported, in comparison with commercially available products, of a novel hybrid system based on silver nanoparticles synthesized using a popular antifungal macrocyclic polyene amphotericin B (AmB) acting both as a reducing and stabilizing/capping agent. The synthesis reaction proceeds in an alkaline environment which prevents aggregation of AmB itself and promotes nanoparticle formation. The innovative approach produces monodisperse (PDI=0.05), AmB-coated silver nanoparticles (AmB-AgNPs) with the diameter ~ 7 nm. The products were characterized using imaging (electron microscopy) and spectroscopic (UV–vis and infrared absorption, dynamic light scattering and Raman scattering) methods. The nanoparticles were tested against Candida albicans, Aspergillus niger and Fusarium culmorum species. For cytotoxicity studies CCD-841CoTr and THP-1 cell lines were used. Particularly high antifungal activity of AmB-AgNPs is interpreted as the result of synergy between the antifungal activity of amphotericin B and silver antimicrobial properties (Ag+ ions release).
No preview · Article · Jan 2016 · Nanomedicine Nanotechnology Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: An idea of a photo-thermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of excited pigment-protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll a and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nano-thermography.
[Show abstract][Hide abstract] ABSTRACT: The precise imaging of biomolecular entities contributes to an understanding of the relationship between their structure and function. However, the resolution of conventional infrared microscopic imaging is diffraction limited and does not exceed a few micrometres. Atomic force microscopy, on the other hand, can detect infrared absorption down to the sub-micrometer level. In the present report, we demonstrate that for multi-bilayer lipid samples containing the plant photosynthetic pigment-protein complex LHCII, the resolution of this latter technique can be better than 20 nm. Such a high resolution is attributable to two factors: (i) the relatively high infrared absorption by the complex that is integrated perpendicular to the plane of the multilayer film, and (ii) the distinctly different mechanical properties and thermal conductivity of the lipid and protein components of the sample.
[Show abstract][Hide abstract] ABSTRACT: The process of primary electric charge separation in photosynthesis takes place in the reaction centers but photosynthesis can operate efficiently and fluently due to the activity of several pigment-protein complexes called antenna, which absorb light quanta and transfer electronic excitations towards the reaction centers. LHCII is the major photosynthetic pigment-protein antenna complex of plants and appears in the trimeric form. Several recent reports point to trimeric organization of LHCII as a key factor responsible for the chloroplast architecture via stabilization of granal organization of the thylakoid membranes. In the present work we address the question: whether such an organization could also directly influence the antenna properties of this pigment-protein complex? Chlorophyll fluorescence analysis reveals that excitation energy transfer in LHCII is substantially more efficient in trimers and dissipative energy losses are higher in monomers. It could be concluded that trimers are exceptionally well suited to perform the antenna function. Possibility of fine regulation of the photosynthetic antenna function via the LHCII trimer-monomer transition is also discussed, based on the fluorescence lifetime analysis in a single chloroplast.
Full-text · Article · Jun 2015 · The Journal of Physical Chemistry B
[Show abstract][Hide abstract] ABSTRACT: Amphotericin B (AmB) is a lifesaving antibiotic used to treat deep-seated mycotic infections. Both the pharmaceutical activity and highly toxic side effects of the drug rely on its interaction with biomembranes, which is governed by the molecular organization of AmB. In the present work we present detailed analysis of self-assembly of AmB molecules in different environments, interesting from the physiological standpoint, based on molecular spectroscopy techniques: electronic absorption, circular dichroism, steady state and time-resolved fluorescence and molecular dynamic calculations. The results show that in the water medium, AmB self-associates to dimeric structures, referred to as "parallel" and "antiparallel". AmB dimers can further assembly into tetramers which can play a role of transmembrane ion channels, affecting electrophysiological homeostasis of a living cell. Understanding structural determinants of self-assembly of AmB opens a way to engineering preparations of the drug which retain pharmaceutical effectiveness under reduced toxicity.
No preview · Article · Nov 2014 · The Journal of Physical Chemistry B
[Show abstract][Hide abstract] ABSTRACT: Bovine serum albumin (BSA) protected nanoclusters (Au and Ag) represent a group of nanomaterials that holds great promise in biophysical applications due to their unique fluorescence properties and lack of toxicity. These metal nanoclusters have utility in a variety of disciplines including catalysis, biosensing, photonics, imaging and molecular electronics. However, they suffer from several disadvantages such as low fluorescence quantum efficiency (typically near 6%) and broad emission spectrum (540 nm to 800 nm). We describe an approach to enhance the apparent brightness of BSA Au clusters by linking them with a high extinction donor organic dye pacific blue (PB). In this conjugate PB acts as a donor to BSA Au clusters and enhances its brightness by resonance energy transfer (RET). We found that the emission of BSA Au clusters can be enhanced by a magnitude of two-fold by resonance energy transfer (RET) from the high extinction donor PB, and BSA Au clusters can act as an acceptor to nanosecond lifetime organic dyes. By pumping the BSA Au clusters using a high extinction donor, one can increase the effective brightness of less bright fluorophores like BSA Au clusters. Moreover, we prepared another conjugate of BSA Au clusters with the near infrared (NIR) dye Dylight 750 (Dy750), where BSA Au clusters act as a donor to Dy750. We observed that BSA Au clusters can function as a donor, showing 46% transfer efficiency to the NIR dye Dy750 with a long lifetime component in the acceptor decay through RET. Such RET-based probes can be used to prevent the problems of a broad emission spectrum associated with the BSA Au clusters. Moreover, transferring energy from BSA Au clusters to Dy750 will result in a RET probe with a narrow emission spectrum and long lifetime component which can be utilized in imaging applications.
[Show abstract][Hide abstract] ABSTRACT: Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes.
[Show abstract][Hide abstract] ABSTRACT: In this report, we investigated the so-called plasmonic platforms prepared to target ultra-short fluorescence and accurate instrumental response function in a time-domain spectroscopy and microscopy. The interaction of metallic nanoparticles with nearby fluorophores results in the increase of the dye fluorescence quantum yield, photostability and decrease of the lifetime parameter. The mentioned properties of platforms were applied to achieve a picosecond fluorescence lifetime (21 ps) of erythrosin B, used later as a better choice for deconvolution of fluorescence decays measured with “color” sensitive photo-detectors. The ultra-short fluorescence standard based on combination of thin layers of silver film, silver colloidal nanoparticles (about 60 nm in diameter), and top layer of erythrosin B embedded in 0.2 % poly(vinyl) alcohol. The response functions were monitored on two photo-detectors; microchannel plate photomultiplier and single photon avalanche photodiode as a Rayleigh scattering and ultra-short fluorescence. We demonstrated that use of the plasmonic base fluorescence standard as an instrumental response function results in the absence of systematic error in lifetime measurements and analysis.
Full-text · Article · Jun 2013 · Journal of Nanoparticle Research
[Show abstract][Hide abstract] ABSTRACT: In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.
[Show abstract][Hide abstract] ABSTRACT: Muscle contraction is brought about by the coupling of chemical energy of ATP hydrolysis to conformational changes in myosin. The chemical changes in myosin are well understood, but the corresponding conformational changes occurring ex-vivo are not. In order to obtain information about conformation it is essential to observe only a few cross-bridges. Observing a large number averages out individual contributions making it impossible to extract kinetic information under steady-state conditions. To minimize the number of observed cross-bridges, only one in 60,000 lever arms in myofibrils were labeled with fluorescent myosin essential light chain 1 (LC1). When such sparsely labeled myofibril is observed through a small aperture of a confocal microscope, ~8 fluorescent lever arms are observed in a volume smaller than a single half-sarcomere. Conformation was measured by recording normalized differences between parallel and perpendicular components of the fluorescence of LC1 (polarized fluorescence, PF). We measured in a single half-sarcomere during isometric contraction ex-vivo, the rate of rotational change of the lever arms and the probability distributions of their orientations. Isometric contraction involved extrusion of massive amount of solvent from the myofilament space, necessitating normalization of PF data. The results indicated that during isometric contraction lever arms rotated at the rate of ~10 s-1 and that the probability distributions of PF were narrower during contraction than relaxation suggesting that the lever arms in contracting muscle were not completely disorganized.
[Show abstract][Hide abstract] ABSTRACT: Previous observations made by our laboratory indicate that the presence of anti-IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury / acute respiratory distress syndrome (ALI/ARDS) is an important prognostic indicator of the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display pro-inflammatory activity towards neutrophils through engagement of FcγRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our current study was to investigate effect of lipopolysaccharide (LPS) on expression of FcγRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in expression of FcγRIIa on the neutrophil surface, and this leads to shortening of the molecular distance between FcγRIIa and TLR4. When such neutrophils are stimulated with anti-IL-8:IL-8 complexes the TLR4 cascade becomes activated via the engagement of FcγRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton's tyrosine kinase (Btk). In conclusion, our study reveals existence of Btk dependent molecular cooperation between FcγRIIa and TLR4 signaling cascades in LPS "primed" human neutrophils. Furthermore, we have used FRET (Fluorescence Lifetime Imaging) to study the interaction between TLR4 and FcγRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcγRIIa.
Full-text · Article · Dec 2012 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment-protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77 K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700 nm, relative to the principal band at 680 nm. The average chlorophyll a fluorescence lifetime at 700 nm changes drastically upon temperature decrease: from 1.04 ns at 300 K to 3.63 ns at 77 K. The results of the experiments lead us to conclude that: (i) the 700 nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.
Full-text · Article · Dec 2012 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Amphotericin B (AmB) is a lifesaving polyene antibiotic used widely to treat deep-seated mycoses. Both the pharmaceutical effectiveness as well as toxic side effects depend on molecular organization of the drug. In the present study, we analyzed steady-state fluorescence, fluorescence anisotropy spectra, fluorescence lifetimes, and fluorescence anisotropy decays of AmB in the systems believed to ensure monomeric organization of the drug and in model lipid membranes. The results of the analyses show that in all of the systems studied, the drug appears in, at least, two spectral forms, interpreted as monomeric and aggregated. Spectroscopic and fluorescence lifetime characteristics of both forms are provided. Interpretation of the fluorescence anisotropy spectra of AmB incorporated into liposomes formed with dipalmitoylphosphatidylcholine let us conclude that monomers of the drug are more tightly bound to the lipid membranes as compared to the aggregates and that AmB aggregates destabilize the membrane structure. Structural model analysis, compared to the analysis of spectral shifts, leads to the conclusion that basic constituents of AmB aggregated structure is a tetramer composed of two hydrogen-bond-stabilized dimers, each dimer formed by molecules twisted by ca. 170°. The tetramer itself can span lipid bilayers and can act as a transmembrane ion channel. Specific aggregate formation of AmB has been concluded as a universal and ubiquitous form of molecular organization of the drug. This process is discussed in terms of toxic side effects of AmB.
[Show abstract][Hide abstract] ABSTRACT: Amphotericin B (AmB) is a lifesaving polyene antibiotic used widely to treat deep-seated mycoses. Both the pharmaceutical effectiveness as well as toxic side effects depend on molecular organization of the drug. In the present study we analyzed steady-state fluorescence, fluorescence anisotropy spectra, fluorescence lifetimes and fluorescence anisotropy decays of AmB in the systems believed to assure monomeric organization of the drug and in model lipid membranes. The results of the analyses show that in all of the systems studied, the drug appears in, at least, two spectral forms, interpreted as monomeric and aggregated. Spectroscopic and fluorescence lifetime characteristics of both forms are provided. Interpretation of the fluorescence anisotropy spectra of AmB incorporated into liposomes formed with dipalmitoylphosphatidylcholine let conclude that monomers of the drug are more tightly bound to the lipid membranes as compared to the aggregates and that AmB aggregates destabilize the membrane structure. Structural model analysis, compared to the analysis of spectral shifts, leads to the conclusion that basic constituents of AmB aggregated structure is a tetramer composed of two hydrogen-bond-stabilized dimers, each dimer formed by molecules twisted by ca. 170 deg. The tetramer itself can span lipid bilayers and can act as a transmembrane ion channel. Specific aggregate formation of AmB has been concluded as an universal and ubiquitous form of molecular organization of the drug. This process is discussed in terms of toxic side effects of AmB.
[Show abstract][Hide abstract] ABSTRACT: Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.
No preview · Article · Mar 2012 · Analytical Biochemistry