RESUMO
C-phycocyanin (C-PC) is the main component of water-soluble light-harvesting complexes (phycobilisomes, PBS) of cyanobacteria. PBS are involved in the absorption of quantum energy and the transfer of electronic excitation energy to the photosystems. A specific environment of C-PC chromophoric groups is provided by the protein matrix structure including protein-protein contacts between different subunits. Registration of C-PC spectral characteristics and the fluorescence anisotropy decay have revealed a significant pH influence on the chromophore microenvironment: at pH 5.0, a chromophore is more significantly interacts with the solvent, whereas at pH 9.0 the chromophore microenvironment becomes more viscous. Conformations of chromophores and the C-PC protein matrix have been studied by Raman and infrared spectroscopy. A decrease in the medium pH results in changes in the secondary structure either the C-PC apoproteins and chromophores, the last one adopts a more folded conformation.
Assuntos
Proteínas de Bactérias , Complexos de Proteínas Captadores de Luz , Ficocianina , Spirulina , Ficocianina/química , Concentração de Íons de Hidrogênio , Polarização de Fluorescência , Análise Espectral Raman , Espectrofotometria Infravermelho , Estrutura Secundária de Proteína , Complexos de Proteínas Captadores de Luz/química , Dobramento de Proteína , Spirulina/enzimologia , Proteínas de Bactérias/químicaRESUMO
Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro.
Assuntos
Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Humanos , Imunoterapia Adotiva/métodos , Linfócitos T , Citocinas/metabolismo , Antineoplásicos/metabolismo , Antígenos CD19/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Complexo CD3/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Artificial biomaterials can significantly increase the rate of tissue regeneration. However, implantation of scaffolds leads not only to accelerated tissue healing but also to an immune response of the organism, which results in the degradation of the biomaterial. The synergy of the immune response and scaffold degradation processes largely determines the efficiency of tissue regeneration. Still, methods suitable for fast, accurate and non-invasive characterization of the degradation degree of biomaterial are highly demandable. Here we show the possibility of monitoring the degradation of decellularized bovine pericardium scaffolds under conditions mimicking the immune response and oxidation processes using multiphoton tomography combined with fluorescence lifetime imaging (MPT-FLIM). We found that the fluorescence lifetimes of genipin-induced cross-links in collagen and oxidation products of collagen are prominent markers of oxidative degradation of scaffolds. This was verified in model experiments, where the oxidation was induced with hypochlorous acid or by exposure to activated neutrophils. The fluorescence decay parameters also correlated with the changes of micromechanical properties of the scaffolds as assessed using atomic force microscopy (AFM). Our results suggest that FLIM can be used for quantitative assessments of the properties and degradation of the scaffolds essential for the wound healing processes in vivo.
Assuntos
Materiais Biocompatíveis , Colágeno , Animais , Materiais Biocompatíveis/farmacologia , Bovinos , Colágeno/metabolismo , Imagem Óptica , Pericárdio/metabolismo , Alicerces TeciduaisRESUMO
Antimicrobial therapy, especially inactivation of multi-antibiotic-resistant strains, requires creating new approaches for drug action and targeted delivery in different environmental conditions. In this work, detonation nanodiamonds (DNDs) were used to deliver polycationic zinc phthalocyanines to E. coli cells. It is shown that in aqueous solutions, zinc phthalocyanines with cholinyl peripheral substituents form complexes with negatively charged DND based on electrostatic interactions. About 40-70 phthalocyanine molecules can bind to a single DND particle, depending on the number of charged groups of the dye molecule. During the complex formation, quenching of phthalocyanine fluorescence and a decrease in its ability to generate reactive oxygen species were observed. In the presence of bacterial cells, phthalocyanine left the complex and induced a photodynamic effect, the magnitude of which depended on the phthalocyanine charge, the molarity of the buffer solution, and the stoichiometry of the phthalocyanine-DND complex. It was found that at physiological values of the ionic strength of the solution, the photodynamic effect of phthalocyanine with a charge of 8+ in combination with a DND is higher than that of the initial phthalocyanine. Thus, nanodiamonds are a promising platform for the delivery of photosensitizers in antimicrobial therapy.
Assuntos
Nanodiamantes , Fotoquimioterapia , Escherichia coli , Indóis/farmacologia , Isoindóis , Fármacos Fotossensibilizantes/farmacologiaRESUMO
CAR-T cell therapy is the most advanced way to treat therapy resistant hematologic cancers, in particular B cell lymphomas and leukemias, with high efficiency. Donor T cells equipped ex vivo with chimeric receptor recognize target tumor cells and kill them using lytic granules. CAR-T cells that recognize CD19 marker of B cells (CD19 CAR-T) are considered the gold standard of CAR-T therapy and are approved by FDA. But in some cases, CD19 CAR-T cell therapy fails due to immune suppressive microenvironment. It is shown that tumor cells upregulate expression of PD-L1 surface molecule that binds and increases level and signal provided by PD-1 receptor on the surface of therapeutic CAR-T cells. Induction of this negative signaling results in functional impairment of cytotoxic program in CAR-T cells. Multiple attempts were made to block PD-1 signaling by reducing binding or surface level of PD-1 in CAR-T cells by various means. In this study we co-expressed CD19-CAR with PD-1-specific VHH domain of anti-PD-1 nanobody to block PD-1/PD-L1 signaling in CD19 CAR-T cells. Unexpectedly, despite increased activation of CAR-T cells with low level of PD-1, these T cells had reduced survival and diminished cytotoxicity. Functional impairment caused by disrupted PD-1 signaling was accompanied by faster maturation and upregulation of exhaustion marker TIGIT in CAR-T cells. We conclude that PD-1 in addition to its direct negative effect on CAR-induced signaling is required for attenuation of strong stimulation leading to cell death and functional exhaustion. These observations suggest that PD-1 downregulation should not be considered as the way to improve the quality of therapeutic CAR-T cells.
RESUMO
Increasing the efficiency of the photodynamic action of the dyes used in photodynamic therapy is crucial in the field of modern biomedicine. There are two main approaches used to increase the efficiency of photosensitizers. The first one is targeted delivery to the object of photodynamic action, while the second one is increasing the absorption capacity of the molecule. Both approaches can be implemented by producing dye-nanoparticle conjugates. In this review, we focus on the features of the latter approach, when nanoparticles act as a light-harvesting agent and nonradiatively transfer the electronic excitation energy to a photosensitizer molecule. We will consider the hybrid photosensitizer-quantum dot complexes with energy transfer occurring according to the inductive-resonance mechanism as an example. The principle consisting in optimizing the design of hybrid complexes is proposed after an analysis of the published data; the parameters affecting the efficiency of energy transfer and the generation of reactive oxygen species in such systems are described.
RESUMO
PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.
Assuntos
Temperatura Baixa , Esterases/antagonistas & inibidores , Esterases/isolamento & purificação , Biblioteca Gênica , Metagenoma/genética , Pergelissolo/microbiologia , Estabilidade Enzimática , Esterases/química , Esterases/metabolismoRESUMO
Enhancement of optical properties of photosensitizers by additional light-harvesting antennas is promising for the improvement of the photodynamic therapy. However, large number of parameters determine interactions of nanoparticles and photosensitizers in complex and, thus the photodynamic efficacy of the hybrid structure. In order to achieve high efficiency of energetic coupling and photodynamic activity of such complexes it is important to know the location of the photosensitizer molecule on the nanoparticle, because it affects the spectral properties of the photosensitizer and the stability of the hybrid complex in vitro/in vivo. In this work complexes of polycationic aluminum phthalocyanines and CdSe/ZnS quantum dots were obtained. We used quantum dots which outer shell consists of polymer with carboxyl groups and provides water solubility and the negative charge of the nanoparticle. We found that phthalocyanine molecules could penetrate deeply into the polymer shell of quantum dot, leading thereby to significant changes in the spectral and photodynamic properties of phthalocyanines. We also showed that noncovalent interactions between phthalocyanine and quantum dot provide possibility for a release of the phthalocyanine from the hybrid complex and its binding to both Gram-positive and Gram-negative bacterial cells. Also, detailed characterization of the nanoparticle core and shell sizes was carried out.
Assuntos
Portadores de Fármacos/química , Indóis/química , Compostos Organometálicos/química , Pontos Quânticos/química , Compostos de Cádmio/química , Transferência Ressonante de Energia de Fluorescência , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Indóis/farmacologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Compostos Organometálicos/farmacologia , Compostos de Selênio/química , Oxigênio Singlete/química , Oxigênio Singlete/metabolismo , Sulfetos/química , Compostos de Zinco/químicaRESUMO
The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.
Assuntos
Proteínas de Bactérias/química , Carotenoides/química , Ficobilissomas/química , Synechocystis/química , Proteínas de Bactérias/genética , Carotenoides/efeitos da radiação , Cristalografia por Raios X , Cinética , Lasers , Luz , Modelos Moleculares , Ficobilissomas/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Espectrometria de Fluorescência , Synechocystis/genéticaRESUMO
We addressed the possibility of using tyrosine (Tyr) fluorescence for monitoring conformational changes of proteins which are undetectable via tryptophan (Trp) fluorescence. The model objects, human (HSA) and bovine (BSA) serum albumins, contain one and two Trp residues, respectively, while Tyr is more uniformly distributed over their structure. The results of the investigation of albumins interaction with ethanol using intrinsic Trp and Tyr steady-state and time-resolved picosecond fluorescence indicated the presence of an intermediate at 10% (v/v) of ethanol in solution, that was supported by the results of extrinsic fluorescence measurements with the Nile Red dye. Based on the comparison of HSA and BSA Trp and Tyr fluorescence, it was suggested that conformational changes at low ethanol concentration are located in the domain III of albumins, which lacks tryptophan residues. The sensitivity of Tyr fluorescence to domain III alterations was further verified by studying albumins interaction with GdnHCl.
Assuntos
Corantes Fluorescentes/química , Soroalbumina Bovina/química , Albumina Sérica/química , Tirosina/química , Animais , Bovinos , Dicroísmo Circular , Fluorescência , Humanos , Nefelometria e Turbidimetria , Oxazinas/química , Domínios Proteicos , Triptofano/químicaRESUMO
Photoprotective mechanisms of cyanobacteria are characterized by several features associated with the structure of their water-soluble antenna complexes - the phycobilisomes (PBs). During energy transfer from PBs to chlorophyll of photosystem reaction centers, the "energy funnel" principle is realized, which regulates energy flux due to the specialized interaction of the PBs core with a quenching molecule capable of effectively dissipating electron excitation energy into heat. The role of the quencher is performed by ketocarotenoid within the photoactive orange carotenoid protein (OCP), which is also a sensor for light flux. At a high level of insolation, OCP is reversibly photoactivated, and this is accompanied by a significant change in its structure and spectral characteristics. Such conformational changes open the possibility for protein-protein interactions between OCP and the PBs core (i.e., activation of photoprotection mechanisms) or the fluorescence recovery protein. Even though OCP was discovered in 1981, little was known about the conformation of its active form until recently, as well as about the properties of homologs of its N and C domains. Studies carried out during recent years have made a breakthrough in understanding of the structural-functional organization of OCP and have enabled discovery of new aspects of the regulation of photoprotection processes in cyanobacteria. This review focuses on aspects of protein-protein interactions between the main participants of photoprotection reactions and on certain properties of representatives of newly discovered families of OCP homologs.
Assuntos
Cianobactérias/fisiologia , Transferência de Energia , Proteínas de Bactérias/química , Ficobilissomas/fisiologiaRESUMO
In direct experiments, rate constants of photochemical (kP) and non-photochemical (kP(+)) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the "dark" reaction center (RC) state, antenna fluorescence decay kinetics were measured under low-intensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a "closed" [P680(+)QA(-)] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680(+)QA(-)] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680(+)) state was found to be ~1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ1 obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680(+)Pheo(-) charge recombination. Thus, for the first time the ratio of kP(+)/kP = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.
Assuntos
Radicais Livres/química , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/metabolismo , Cátions/química , Ditionita/química , Cinética , Complexo de Proteína do Fotossistema II/química , Folhas de Planta/metabolismo , Espectrometria de FluorescênciaRESUMO
Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cromatografia em Gel , Clonagem Molecular , Cianobactérias/fisiologia , Fluorescência , Polarização de Fluorescência , Fluorometria , Análise Espectral Raman , Synechocystis/fisiologiaRESUMO
In a direct experiment, the rate constants of photochemical k p and non-photochemical k p+ quenching of the chlorophyll fluorescence have been determined in spinach photosystem II (PS II) membrane fragments, oxygen-evolving PS II core, as well as manganese-depleted PS II particles using pulse fluorimetry. In the dark-adapted reaction center(s) (RC), the fluorescence decay kinetics of the antenna were measured at low-intensity picosecond pulsed excitation. To create a "closed" P680+Q A- state, RCs were illuminated by high-intensity actinic flash 8 ns prior to the measuring flash. The obtained data were approximated by the sum of two decaying exponents. It was found that the antennae fluorescence quenching efficiency by the oxidized photoactive pigment of RC P680+ was about 1.5 times higher than that of the neutral P680 state. These results were confirmed by a single-photon counting technique, which allowed to resolve the additional slow component of the fluorescence decay. Slow component was assigned to the charge recombination of P680+Pheo- in PS II RC. Thus, for the first time, the ratio k p+ /k p â 1.5 was found directly. The mechanism of the higher efficiency of non-photochemical quenching comparing to photochemical quenching is discussed.
Assuntos
Complexo de Proteína do Fotossistema II/metabolismo , Cátions/metabolismo , Clorofila/metabolismo , Fluorescência , Radicais Livres/metabolismo , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Oxigênio/metabolismo , Spinacia oleracea/metabolismoRESUMO
Excitation energy transfer (EET) is observed in hybrid structures that composed of allophycocyanin and CdSe/ZnS core-shell quantum dot (QD). We demonstrate that the EET efficiency in such systems could be significantly increased under conditions inducing monomerization of allophycocyanin trimers. For these purposes, the EET efficiency was estimated under different experimental conditions (pH, high temperature or the presence of NaSCN) for self-assembled hybrid structures. Additionally, the hybrid structures were stabilized by covalent coupling which resulted in approximately 20-fold enhancement of allophycocyanin fluorescence upon excitation of QDs. The observed effect provides new opportunities for the practical implementation of hybrid systems as fluorescent markers.
Assuntos
Ficocianina/química , Pontos Quânticos , Transferência de Energia , Concentração de Íons de Hidrogênio , Estrutura Molecular , TemperaturaRESUMO
Semiconductor nanocrystals (CdSe/ZnS quantum dots, QDs) were used as inorganic focusing antenna, allowing for the enhancement of fluorescence and photosensitizing activity of polycationic aluminum phthalocyanines (PCs). It was found that QDs form stable complexes with PCs in aqueous solutions due to electrostatic interactions. In such hybrid complexes, we observed highly efficient nonradiative energy transfer from QD to PC, leading to a sharp increase in the effective absorption cross section of PC in the absorption bands of the CdSe/ZnS quantum dots. When hybrid complexes are excited within these bands, the intensity of PC fluorescence and the rate of photosensitized singlet oxygen generation increases significantly (up to 500 and 350%, correspondingly) compared to free PC at the same concentration. The observed effect is of interest for modeling primary stages of photosynthesis and increasing photosensitizing activity of dyes used in photodynamic therapy.
Assuntos
Indóis/química , Nanoestruturas/química , Compostos Organometálicos/química , Fármacos Fotossensibilizantes/química , Pontos Quânticos/química , Transferência de Energia , Fluorescência , Fotoquimioterapia/instrumentação , Fármacos Fotossensibilizantes/síntese químicaRESUMO
Under high photon flux density of solar radiation, the photosynthetic apparatus can be damaged. To prevent this photodestruction, cyanobacteria developed special mechanisms of non-photochemical quenching (NPQ) of excitation energy in phycobilisomes. In Synechocystis, NPQ is triggered by the orange carotenoid protein (OCP), which is sensitive to blue-green illumination allowing it to bind to the phycobilisome reducing the flow of energy to the photosystems. Consequent decoupling of OCP and recovery of phycobilisome fluorescence in vivo is controlled by the so called fluorescence recovery protein (FRP). In this work, the role of the phycobilisome core components, apcD and apcF, in non-photochemical quenching and subsequent fluorescence recovery in the phycobilisomes of the cyanobacterium Synechocystis sp. PCC6803 has been investigated. Using a single photon counting technique, we have registered fluorescence decay spectra with picosecond time resolution during fluorescence recovery. In order to estimate the activation energy for the photocycle, spectroscopic studies in dependency on the temperature from 5 to 45 °C have been performed. It was found that fluorescence quenching and recovery were strongly temperature dependent for all strains exhibiting characteristic non-linear time courses. The rise of the fluorescence intensity during fluorescence recovery after NPQ can be completely described by the increase of the phycobilisome core fluorescence lifetime. It was shown that fluorescence recovery of apcD- and apcF-deficient mutants is characterized by a significantly lower activation energy barrier compared to wild type. This phenomenon indicates that apcD and apcF gene products may be required for proper interaction of FRP and OCP coupled to the phycobilisome core. In addition, we found that the rate of fluorescence recovery decreases with an increase of the non-photochemical quenching amplitude, probably due to depletion of substrate for the enzymatic reaction catalyzed by FRP.
Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Ficobilissomas/metabolismo , Synechocystis/metabolismo , Fluorescência , Luz , Ficobilissomas/efeitos da radiação , Synechocystis/efeitos da radiação , TemperaturaRESUMO
Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.
Assuntos
Líquens/citologia , Microscopia de Fluorescência/métodos , Clorofila/metabolismo , Dessecação , Fluorescência , Cinética , Líquens/fisiologia , Complexo de Proteína do Fotossistema II/fisiologiaRESUMO
As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures - from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 µmolphotonsm⻲s⻹. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Assuntos
Fluorometria/métodos , Ficobilissomas/química , Synechocystis/metabolismo , Transferência de Energia , Fluorescência , Conformação ProteicaRESUMO
We show that semiconductor nanocrystals (quantum dots, QD) can be used to increase the absorption capacity of pigment-protein complexes. In a mixture of photosystem 2 core complex (PS2) and QD, the fluorescence of the latter decreases several-fold due to the transfer of the absorbed energy to the PS2 core complex. We discuss Förster's inductive-resonance mechanism as a possible way of energy transfer in donor-acceptor pairs QD-PS2 core complex. Calculations based on the experimental data show that the enhancement of PS2 fluorescence and the rate of Q(A) reduction increase up to 60% due to efficient energy migration from QD to PS2.