RESUMO
The widespread use of disinfectants and antiseptics, and consequently their release into the environment, determines the relevance of studying their potential impact on the main producers of organic matter on the planet-photosynthetic organisms. The review examines the effects of some biguanides and quaternary ammonium compounds, octenidine, miramistin, chlorhexidine, and picloxidine, on the functioning of the photosynthetic apparatus of various organisms (Strakhovskaya et al. in Photosynth Res 147:197-209, 2021; Knox et al. in Photosynth Res 153:103, 2022; Paschenko et al. in Photosynth Res 155:93-105, 2023a, Photosynth Res 2023b). A common feature of these antiseptics is the combination of hydrophobic and hydrophilic regions in the molecules, the latter carrying a positive charge(s). The comparison of the results obtained with intact bacterial membrane vesicles (chromatophores) and purified pigment-protein complexes (photosystem II and I) of oxygenic organisms allows us to draw conclusions about the mechanisms of the cationic antiseptic action on the functional properties of the components of the photosynthetic apparatus.
Assuntos
Anti-Infecciosos Locais , Fotossíntese , Fotossíntese/efeitos dos fármacos , Anti-Infecciosos Locais/farmacologia , Luz , Cátions , Compostos de Amônio Quaternário/farmacologia , Compostos de Amônio Quaternário/químicaRESUMO
In this study, the effects of cationic antiseptics such as chlorhexidine, picloxidine, miramistin, and octenidine at concentrations up to 150 µM on fluorescence spectra and its lifetimes, as well as on light-induced electron transfer in protein-pigment complexes of photosystem I (PSI) isolated from cyanobacterium Synechocystis sp. PCC 6803 have been studied. In doing so, octenidine turned out to be the most "effective" in terms of its influence on the spectral and functional characteristics of PSI complexes. It has been shown that the rate of energy migration from short-wavelength forms of light-harvesting chlorophyll to long-wavelength ones slows down upon addition of octenidine to the PSI suspension. After photo-separation of charges between the primary electron donor P700 and the terminal iron-sulfur center(s) FA/FB, the rate of forward electron transfer from (FA/FB)- to the external medium slows down while the rate of charge recombination between reduced FA/FB- and photooxidized P700+ increases. The paper considers the possible causes of the observed action of the antiseptic.
Assuntos
Anti-Infecciosos Locais , Iminas , Piridinas , Synechocystis , Complexo de Proteína do Fotossistema I , Elétrons , CátionsRESUMO
Herein, the effect of cationic antiseptics (chlorhexidine, picloxidine, miramistin, octenidine) on the initial processes of the delivery of light energy and its efficient use by the reaction centers in intact spinach photosystem II core complexes has been investigated. The characteristic effects-an increase in the fluorescence yield of light-harvesting pigments and a slowdown in the rate of energy migration in bacterial photosynthetic chromatophores has been recently demonstrated mainly in the presence of octenidine (Strakhovskaya et al., in Photosynth Res 147:197-209, 2021; Knox et al., in Photosynth Res, https://doi.org/10.1007/s11120-022-00909-8 , 2022). In this study, we also observed that in the presence of octenidine, the fluorescence intensity of photosystem II core complexes increases by 5-10 times, and the rate of energy migration from antennae to the reaction centers decreases by 3 times. In addition, with an increase in the concentration of this antiseptic, a new effect related to a shift of the spectrum, absorption and fluorescence to the short-wavelength region has been found. Similar effects were observed when detergent Triton X-100 was added to photosystem II samples. We concluded that the antiseptic primarily affects the structure of the internal light-harvesting antenna (CP43 and CP47), through which the excitation energy is delivered to the reaction center. As a result of such an impact, the chlorophyll molecules in this structure are destabilized and their optical and functional characteristics change.
Assuntos
Anti-Infecciosos Locais , Complexo de Proteína do Fotossistema II , Complexo de Proteína do Fotossistema II/química , Complexos de Proteínas Captadores de Luz/química , Clorofila/química , Espectrometria de FluorescênciaRESUMO
Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1-reaction center core complex and to produce the long-wavelength ("red") shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.
Assuntos
Cromatóforos , Rhodobacter sphaeroides , Rhodobacter sphaeroides/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Bacterioclorofilas/metabolismo , Dipiridamol/farmacologia , Dipiridamol/metabolismo , Transferência de Energia , Proteínas de Membrana/metabolismo , Cromatóforos/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Photosynthetic membrane complexes of purple bacteria are convenient and informative macromolecular systems for studying the mechanisms of action of various physicochemical factors on the functioning of catalytic proteins both in an isolated state and as part of functional membranes. In this work, we studied the effect of cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine) on the fluorescence intensity and the efficiency of energy transfer from the light-harvesting LH1 complex to the reaction center (RC) of Rhodospirillum rubrum chromatophores. The effect of antiseptics on the fluorescence intensity and the energy transfer increased in the following order: chlorhexidine, picloxydine, miramistin, octenidine. The most pronounced changes in the intensity and lifetime of fluorescence were observed with the addition of miramistin and octenidine. At the same concentration of antiseptics, the increase in fluorescence intensity was 2-3 times higher than the increase in its lifetime. It is concluded that the addition of antiseptics decreases the efficiency of the energy migration LH1 â RC and increases the fluorescence rate constant kfl. We associate the latter with a change in the polarization of the microenvironment of bacteriochlorophyll molecules upon the addition of charged antiseptic molecules. A possible mechanism of antiseptic action on R. rubrum chromatophores is considered. This work is a continuation of the study of the effect of antiseptics on the energy transfer and fluorescence intensity in chromatophores of purple bacteria published earlier in Photosynthesis Research (Strakhovskaya et al. in Photosyn Res 147:197-209, 2021).
Assuntos
Anti-Infecciosos Locais , Cromatóforos , Complexo de Proteínas do Centro de Reação Fotossintética , Rhodospirillum rubrum , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Compostos de Benzalcônio , Clorexidina/metabolismo , Cromatóforos/metabolismo , Fluorescência , Iminas , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Piridinas , Rhodospirillum rubrum/metabolismoRESUMO
Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Fotoquímica , Cristalografia , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação ProteicaRESUMO
Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.
Assuntos
Anti-Infecciosos Locais/farmacologia , Cromatóforos Bacterianos/efeitos dos fármacos , Transferência de Energia/efeitos dos fármacos , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos dos fármacos , Rhodobacter sphaeroides/fisiologia , Cardiolipinas/química , Membrana Celular/efeitos dos fármacos , Cinética , Luz , Complexos de Proteínas Captadores de Luz/efeitos dos fármacos , Fosfatidilgliceróis/química , Fotossíntese/efeitos dos fármacos , Rhodobacter sphaeroides/química , Espectrometria de FluorescênciaRESUMO
Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called "Great Oxygenation Event" that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.
Assuntos
Cianobactérias/química , Transferência de Energia , Ficobilissomas/fisiologia , Protetores contra Radiação/química , Origem da Vida , Fotossíntese , Ficobiliproteínas/fisiologia , Radiação Ionizante , Voo EspacialRESUMO
The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the heating of pre-frozen to - 180 °C in the dark or in the light-activated preparations has been studied. Fluorescence lifetime in samples frozen in the light was longer than in samples frozen in the dark. For samples in 65% glycerol at λreg = 335 nm and at 20 °C, the lifetime of components were as follows: τ1 ≈ 1.2 ns, τ2 ≈ 4.9 ns, and τ3 ≈ 20 ns. The contribution of the first component was negligible. To analyze the contribution of components 2 and 3 derived from frozen-thawed samples, two temperature ranges from - 180 to - 90 °C and above - 90 °C are considered. In doing so, the contributions of these components appear antiphase course to each other. The dependence on temperature of these contributions is explained by the influence of the microconformational protein dynamics on the tryptophan fluorescence lifetime. In the present work, a comparative analysis of temperature-dependent conformational dynamics and electron transfer in cyanobacterial PSI (Schlodder et al., in Biochemistry 37:9466-9476, 1998) and Rhodobacter sphaeroides reaction center complexes (Knox et al., in J Photochem Photobiol B 180:140-148, 2018) was also carried out.
Assuntos
Cianobactérias/metabolismo , Fluorescência , Luz , Complexo de Proteína do Fotossistema I/metabolismo , Triptofano/química , Cianobactérias/efeitos da radiação , TemperaturaRESUMO
The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm2) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure-polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC + QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC + QD) complexes.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Pontos Quânticos , Raios Ultravioleta , Calefação , Rhodobacter/metabolismo , Trealose/metabolismoRESUMO
The temperature dependencies of the rate of dark recombination of separated charges between the photoactive bacteriochlorophyll and the primary quinone acceptor (QA) in photosynthetic reaction centers (RCs) of the purple bacteria Rhodobacter sphaeroides (Rb. sphaeroides) were investigated. Measurements were performed in water-glycerol and trehalose environments after freezing to -180⯰C in the dark and under actinic light with subsequent heating. Simultaneously, the RC tryptophanyl fluorescence lifetime in the spectral range between 323 and 348â¯nm was measured under these conditions. A correlation was found between the temperature dependencies of the functional and dynamic parameters of RCs in different solvent mixtures. For the first time, differences in the average fluorescence lifetime of tryptophanyl residues were measured between RCs frozen in the dark and in the actinic light. The obtained results can be explained by the RC transitions between different conformational states and the dynamic processes in the structure of the hydrogen bonds of RCs. We assumed that RCs exist in two main microconformations - "fast" and "slow", which are characterized by different rates of P+ and QA- recombination reactions. The "fast" conformation is induced in frozen RCs in the dark, while the "slow" conformation of RC occurs when the RC preparation is frozen under actinic light. An explanation of the temperature dependencies of tryptophan fluorescence lifetimes in RC proteins was made under the assumption that temperature changes affect mainly the electron transfer from the indole ring of the tryptophan molecule to the nearest amide or carboxyl groups.
Assuntos
Benzoquinonas/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/metabolismo , Triptofano/química , Transporte de Elétrons , Ligação de Hidrogênio , Cinética , Luz , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Teoria Quântica , Espectrometria de Fluorescência , Temperatura , Triptofano/metabolismoRESUMO
In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.
RESUMO
Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.
Assuntos
Apoproteínas/metabolismo , Proteínas de Bactérias/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Apoproteínas/química , Proteínas de Bactérias/química , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cisteína/metabolismo , Difusão , Hidrodinâmica , Espectrometria de Massas , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Coloração e Rotulagem , Compostos de Sulfidrila/metabolismoRESUMO
Orange Carotenoid Protein (OCP) is known as an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for blue-green light absorbing keto-carotenoids, which excitation causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. Although recent studies greatly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced, to our knowledge, a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of photoactive OCP from completely photoinactive species. We accurately analyze the peculiarities of this process that, to the best of our knowledge, appears unique, a previously uncharacterized protein-to-protein carotenoid transfer mechanism. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection.
Assuntos
Apoproteínas/metabolismo , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Apoproteínas/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrodinâmica , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Processos Fotoquímicos , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Synechocystis , Termodinâmica , Difração de Raios XRESUMO
Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.
Assuntos
Resposta ao Choque Frio/fisiologia , Fluidez de Membrana/fisiologia , Synechocystis/fisiologia , Anisotropia , Temperatura Baixa , Ácidos Graxos/metabolismo , Fluorescência , Regulação Bacteriana da Expressão Gênica , Cinética , Lipídeos de Membrana/metabolismo , Oxirredução , Plastoquinona/metabolismo , Análise Espectral Raman , Synechocystis/genéticaRESUMO
Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/química , Fotoperíodo , Modelos Moleculares , Conformação Proteica , Rodaminas/química , Solventes/química , Espectrometria de Fluorescência , Temperatura , ViscosidadeRESUMO
Quantum dots (QDs) absorb ultraviolet and long-wavelength visible light energy much more efficiently than natural bacterial light-harvesting proteins and can transfer the excitation energy to photosynthetic reaction centers (RCs). Inclusion of RCs combined with QDs as antennae into liposomes opens new opportunities for using such hybrid systems as a basis for artificial energy-transforming devices that potentially can operate with greater efficiency and stability than devices based only on biological components or inorganic components alone. RCs from Rhodobacter sphaeroides and QDs (CdSe/ZnS with hydrophilic covering) were embedded in lecithin liposomes by extrusion of a solution of multilayer lipid vesicles through a polycarbonate membrane or by dialysis of lipids and proteins dispersed with excess detergent. The efficiency of RC and QD interaction within the liposomes was estimated using fluorescence excitation spectra of the photoactive bacteriochlorophyll of the RCs and by measuring the fluorescence decay kinetics of the QDs. The functional activity of the RCs in hybrid complexes was fully maintained, and their stability was even increased. The efficiency of energy transfer between QDs and RCs and conditions of long-term stability of function of such hybrid complexes in film preparations were investigated as well. It was found that dry films containing RCs and QDs, maintained at atmospheric humidity, are capable of maintaining their functional activity for at least some months as judged by measurements of their spectral characteristics, efficiency of energy transfer from QDs to RCs and RC electron transport activity. Addition of trehalose to the films increases the stability further, especially for films maintained at low humidity. These stable hybrid film structures are promising for further studies towards developing new phototransformation devices for biotechnological applications.
Assuntos
Lecitinas/química , Lipossomos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteobactérias/metabolismo , Pontos Quânticos , Eletroforese em Gel de Ágar , Microscopia Eletrônica de TransmissãoRESUMO
Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the ß-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.
Assuntos
Proteínas de Bactérias/química , Proteínas Luminescentes/química , Simulação de Dinâmica Molecular , Absorção de Radiação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cianobactérias/química , Corantes Fluorescentes/farmacologia , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.