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Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.
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VIH-1/metabolismo , Virión/metabolismo , Ensamble de Virus/fisiología , Línea Celular , Membrana Celular/metabolismo , Humanos , Liberación del Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The concept of effective one-electron potentials (EOPs) has proven to be extremely useful in efficient description of electronic structure of chemical systems, especially extended molecular aggregates such as interacting molecules in condensed phases. Here, a general method for EOP-based elimination of electron repulsion integrals is presented, that is tuned toward the fragment-based calculation methodologies such as the second generation of the effective fragment potentials (EFP2) method. Two general types of the EOP operator matrix elements are distinguished and treated either via the distributed multipole expansion or the extended density fitting (DF) schemes developed in this work. The EOP technique is then applied to reduce the high computational costs of the effective fragment charge-transfer (CT) terms being the bottleneck of EFP2 potentials. The alternative EOP-based CT energy model is proposed, derived within the framework of intermolecular perturbation theory with Hartree-Fock noninteracting reference wavefunctions, compatible with the original EFP2 formulation. It is found that the computational cost of the EFP2 total interaction energy calculation can be reduced by up to 38 times when using the EOP-based formulation of CT energy, as compared to the original EFP2 scheme, without compromising the accuracy for a wide range of weakly interacting neutral and ionic molecular fragments. The proposed model can thus be used routinely within the EFP2 framework.
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Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.
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Dinamina II/genética , Endocitosis/genética , Mutación/genética , Miopatías Estructurales Congénitas/genética , Adulto , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Clatrina/genética , Femenino , Fibroblastos/patología , Humanos , Cinética , Masculino , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Microscopía Fluorescente/métodosRESUMEN
It emerges from numerous experiments that LHCII, the major photosynthetic antenna complex of plants, can appear not only in the trimeric or monomeric states but also as a dimer. We address the problem whether the dimeric form of the complex is just a simple intermediate element of the trimer-monomer transformation or if it can also be a physiologically relevant molecular organization form? Dimers of LHCII were analyzed with application of native electrophoresis, time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy. The results reveal the appearance of two types of LHCII dimers: one formed by the dissociation of one monomer from the trimeric structure and the other formed by association of monomers into a distinctively different molecular organizational form, characterized by a high rate of chlorophyll excitation quenching. The hypothetical structure of such an energy quencher is proposed. The high light-induced LHCII dimerization is discussed as a potential element of the photoprotective response in plants.
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Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismo , Clorofila/metabolismo , Luz , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/química , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Spinacia oleracea/metabolismo , Spinacia oleracea/efectos de la radiaciónRESUMEN
This communication presents a structure-property study of a few novel pyridine-based difluoroborate compounds with a N-BF2-O core, which exhibit outstanding fluorescence properties. To exploit their potential for two-photon bioimaging, relationships between the two-photon action cross section and systematic structural modifications have been investigated and unravelled.
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We have examined several approaches relying on the Polarizable Embedding (PE) scheme to predict optical band shapes for two chalcone molecules in methanol solution. The PE-TDDFT and PERI-CC2 methods were combined with molecular dynamics simulations, where the solute geometry was kept either as rigid, flexible or partly-flexible (restrained) body. The first approach, termed RBMD-PE-TDDFT, was employed to estimate the inhomogeneous broadening for subsequent convolution with the vibrationally-resolved spectra of the molecule in solution determined quantum-mechanically (QM). As demonstrated, the RBMD-PE-TDDFT/QM-PCM approach delivers accurate band widths, also reproducing their correct asymmetric shapes. Further refinement can be obtained by the estimation of the inhomogeneous broadening using the RBMD-PERI-CC2 method. On the other hand, the remaining two approaches (FBMD-PE-TDDFT and ResBMD-PE-TDDFT), which lack quantum-mechanical treatment of molecular vibrations, lead to underestimated band widths. In this study, we also proposed a simple strategy regarding the rapid selection of the exchange-correlation functional for the simulations of vibrationally-resolved one- and two-photon absorption spectra based on two easy-to-compute metrics.
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Chalconas/química , Simulación de Dinámica Molecular , Estructura Molecular , Fotones , Relación Estructura-Actividad Cuantitativa , Teoría Cuántica , Soluciones/química , VibraciónRESUMEN
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.
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Luz , Estrés Fisiológico , Tilacoides/química , Tilacoides/efectos de la radiación , Galactolípidos/química , Immunoblotting , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/ultraestructura , Lípidos de la Membrana/química , Membranas Artificiales , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Moleculares , Fosforilación/efectos de la radiación , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Conformación Proteica , Espectrofotometría Infrarroja , Spinacia oleracea/química , Spinacia oleracea/metabolismo , Spinacia oleracea/efectos de la radiación , Tilacoides/ultraestructura , Difracción de Rayos X , Xantófilas/química , ZeaxantinasRESUMEN
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.
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Complejos de Proteína Captadores de Luz/química , Simulación del Acoplamiento Molecular , Complejo de Proteína del Fotosistema II/química , Zeaxantinas/química , Sitios de Unión/efectos de la radiación , Luz , Complejos de Proteína Captadores de Luz/efectos de la radiación , Complejo de Proteína del Fotosistema II/efectos de la radiación , Unión Proteica/efectos de la radiación , Conformación Proteica/efectos de la radiación , Dosis de Radiación , Xantófilas/química , Xantófilas/efectos de la radiación , Zeaxantinas/efectos de la radiaciónRESUMEN
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 77K 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 700nm, relative to the principal band at 680nm. The average chlorophyll a fluorescence lifetime at 700nm changes drastically upon temperature decrease: from 1.04ns at 300K to 3.63ns at 77K. The results of the experiments lead us to conclude that: (i) the 700nm 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.
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Complejos de Proteína Captadores de Luz/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Clorofila/química , Clorofila A , Retroalimentación Fisiológica , Luz , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.
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Imagenología Tridimensional/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/patología , Análisis de la Célula Individual/métodos , Biofisica , Línea Celular Tumoral , Diatomeas/citología , Humanos , Concentración de Iones de Hidrógeno , Melanoma , Microscopía Electrónica de RastreoRESUMEN
The present work investigates the performance of exchange-correlation functionals in the prediction of two-photon absorption (2PA) strengths. For this purpose, we considered six common functionals used for studying 2PA processes and tested these on six organoboron chelates. The set consisted of two semilocal (PBE and BLYP), two hybrid (B3LYP and PBE0), and two range-separated (LC-BLYP and CAM-B3LYP) functionals. The RI-CC2 method was chosen as a reference level and was found to give results consistent with the experimental data that are available for three of the molecules considered. Of the six exchange-correlation functionals studied, only the range-separated functionals predict an ordering of the 2PA strengths that is consistent with experimental and RI-CC2 results. Even though the range-separated functionals predict correct relative trends, the absolute values for the 2PA strengths are underestimated by a factor of 2-6 for the molecules considered. An in-depth analysis, on the basis of the derived generalized few-state model expression for the 2PA strength for a coupled-cluster wave function, reveals that the problem with these functionals can be linked to underestimated excited-state dipole moments and, to a lesser extent, overestimated excitation energies. The semilocal and hybrid functionals exhibit less predictable errors and a variation in the 2PA strengths in disagreement with the reference results. The semilocal and hybrid functionals show smaller average errors than the range-separated functionals, but our analysis reveals that this is due to fortuitous error cancellation between excitation energies and the transition dipole moments. Our results constitute a warning against using currently available exchange-correlation functionals in the prediction of 2PA strengths and highlight the need for functionals that correctly describe the electron density of excited electronic states.
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Diabetes mellitus is an independent cardiovascular risk factor, considered an equivalent of coronary artery disease in terms of prognosis. A history of acute coronary syndrome is a strong predictor of another coronary episode, and cardiovascular complications are the leading cause of mortality in diabetic patients. Many patients with coronary artery disease suffer from concomitant diabetes or pre-diabetes. There are 3 strategies of coronary artery disease treatment: conservative management, coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI). Since drug-eluting stents (DES) were developed, PCI has become one of the most widespread interventional cardiology procedures performed in Europe and worldwide. Among all coronary risk factors, diabetes mellitus remains the most important predictor of unfavorable outcomes of revascularization therapy. This paper reviews the current evidence regarding revascularization in diabetic patients, with particular emphasis on PCI. A systematic analysis of clinical trials of CABG and PCI, especially with DES, was conducted.
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Enfermedad de la Arteria Coronaria/terapia , Complicaciones de la Diabetes/terapia , Revascularización Miocárdica/métodos , Puente de Arteria Coronaria/métodos , Humanos , Intervención Coronaria Percutánea/métodosRESUMEN
This article aims at a quantitative assessment of the performances of a panel of exchange-correlation functionals, including semilocal (BLYP and PBE), global hybrids (B3LYP, PBE0, M06, BHandHLYP, M06-2X, and M06-HF), and range-separated hybrids (CAM-B3LYP, LC-ωPBE, LC-BLYP, ωB97X, and ωB97X-D), in predicting the vibrationally resolved absorption spectra of BF2-carrying compounds. To this end, for 19 difluoroborates as examples, we use, as a metric, the vibrational reorganization energy (λvib) that can be determined based on the computationally efficient linear coupling model (a.k.a. vertical gradient method). The reference values of λvib were determined by employing the CC2 method combined with the cc-pVTZ basis set for a representative subset of molecules. To validate the performances of CC2, comparisons with experimental data have been carried out as well. This study shows that the vibrational reorganization energy, involving Huang-Rhys factors and normal-mode frequencies, can indeed be used to quantify the reliability of functionals in the calculations of the vibrational fine structure of absorption bands, i.e., an accurate prediction of the vibrational reorganization energy leads to absorption band shapes better fitting the selected reference. The CAM-B3LYP, M06-2X, ωB97X-D, ωB97X, and BHandHLYP functionals all deliver vibrational reorganization energies with absolute relative errors smaller than 20% compared to CC2, whereas 10% accuracy can be achieved with the first three functionals. Indeed, the set of examined exchange-correlation functionals can be divided into three groups: (i) BLYP, B3LYP, PBE, PBE0, and M06 yield inaccurate band shapes (λvib,TDDFT < λvib,CC2), (ii) BHandHLYP, CAM-B3LYP, M06-2X, ωB97X, and ωB97X-D provide accurate band shapes (λvib,TDDFT ≈ λvib,CC2), and (iii) LC-ωPBE, LC-BLYP, and M06-HF deliver rather poor band topologies (λvib,TDDFT > λvib,CC2). This study also demonstrates that λvib can be reliably estimated using the CC2 model and the relatively small cc-pVDZ basis set. Therefore, the linear coupling model combined with the CC2/cc-pVDZ level of theory can be used as a very efficient approach to determine λvib values that can be used to select the most adequate functional for more accurate vibronic calculations, e.g., including more refined models and environmental effects.
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The efficient and fluent operation of photosynthesis in plants relies on activity of pigment-protein complexes called antenna, absorbing light and transferring excitations toward the reaction centers. Here we show, based on the results of the fluorescence lifetime imaging analyses of single chloroplasts, that pigment-protein complexes, in dark-adapted plants, are not able to act effectively as photosynthetic antennas, due to pronounced, adverse excitation quenching. It appeared that the antenna function could be activated by a short (on a minute timescale) illumination with light of relatively low intensity, substantially below the photosynthesis saturation threshold. The low-light-induced activation of the antenna function was attributed to phosphorylation of the major accessory light-harvesting complex LHCII, based on the fact that such a mechanism was not observed in the stn7 Arabidopsis thaliana mutant, with impaired LHCII phosphorylation. It is proposed that the protein phosphorylation-controlled change in the LHCII clustering ability provides mechanistic background for this regulatory process.
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Arabidopsis/metabolismo , Cloroplastos/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Fotosíntesis , Microscopía FluorescenteRESUMEN
In this work, we examine, by means of computational methods, the mechanism of Zn(2+) sensing by a bipyridine-centered, D-π-A-π-D-type ratiometric molecular probe. According to recently published experimental data [Divya, K. P.; Sreejith, S.; Ashokkumar, P.; Yuzhan, K.; Peng, Q.; Maji, S. K.; Tong, Y.; Yu, H.; Zhao, Y.; Ramamurthy, P.; Ajayaghosh, A. A ratiometric fluorescent molecular probe with enhanced two-photon response upon Zn(2+) binding for in vitro and in vivo bioimaging. Chem. Sci. 2014, 5, 3469-3474], after coordination to zinc ions the probe exhibits a large enhancement of the two-photon absorption cross section. The goal of our investigation was to elucidate the mechanism behind this phenomenon. For this purpose, linear and nonlinear optical properties of the unbound (cation-free) and bound probe were calculated, including the influence of solute-solvent interactions, implicitly using a polarizable continuum model and explicitely employing the QM/MM approach. Because the results of the calculations indicate that many conformers of the probe are energetically accessible at room temperature in solution and hence contribute to the signal, structure-property relationships were also taken into account. Results of our simulations demonstrate that the one-photon absorption bands for both the unbound and bound forms correspond to the bright π â π* transition to the first excited state, which, on the other hand, exhibits negligible two-photon activity. On the basis of the results of the quadratic response calculations, we put forward a notion that it is the second excited state that gives the strong signal in the experimental nonlinear spectrum. To explain the differences in the two-photon absorption activity for the two lowest-lying excited states and nonlinear response enhancement upon binding, we employed the generalized few-state model including the ground, first, and second excited states. The analysis of the optical channel suggests that the large two-photon response is due to the coordination-induced increase of the transition moment from the first to the second excited state.
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Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans-cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed.
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Complejos de Proteína Captadores de Luz/química , Luz , Proteínas de Plantas/química , Xantófilas/química , Isomerismo , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Complejos de Proteína Captadores de Luz/metabolismo , Microscopía Confocal , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Estructura Cuaternaria de Proteína , Espectrometría Raman , Spinacia oleracea/metabolismo , Xantófilas/aislamiento & purificación , Xantófilas/metabolismoRESUMEN
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 toward 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 of 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.
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Complejo de Proteína del Fotosistema II/química , Cloroplastos/química , Electroforesis , Cinética , Microscopía Fluorescente , Complejo de Proteína del Fotosistema II/aislamiento & purificación , Análisis Espectral , Spinacia oleraceaRESUMEN
We report on the nonlinear optical properties measurements and quantum-chemical calculations of a well-known photochromic system consisting of spiropyran and the merocyanine photoproduct. The study of nonlinear absorption and refraction properties of the molecules dissolved in chloroform were performed with the Z-scan technique, using femtosecond pulses in a wide range of wavelengths. Maxima in the two-photon absorption spectrum at 700 and 1050 nm were found for the merocyanine form, and the corresponding two-photon absorption cross section is 80 GM and 20 GM, respectively. The latter feature does not vanish completely in the nonlinear spectrum of the spiropyran form, possibly because of the existence of some photoconversion caused by the laser beam during the measurements. A nonlinear absorption peak at 900 nm is found in the spiropyran form with an effective cross section of about 20 GM; it is likely due to three-photon absorption or to absorption by some intermediate species. The experimental data are supported by calculations performed with the use of a hybrid quantum mechanics-molecular mechanics approach.