RESUMEN
The Orange Carotenoid Protein (OCP) regulates cyanobacterial photosynthetic activity through photoactivation in intense light. A hydrogen bonding network involving the keto-carotenoid oxygen and Y201 and W288 residues prevents the spontaneous activation of dark-adapted OCP. To investigate the role of the hydrogen bonds in OCP photocycling, we introduced non-canonical amino acids near the keto-carotenoid, particularly iodine at the meta-position of Y201. This modification significantly increased the yield of red OCP photoproducts, albeit with a shorter lifetime. Changes in tryptophan fluorescence during photocycling influenced by the presence of iodine near W288 revealed interactions between Y201 and W288 in the absence of the carotenoid in the C-domain. We propose that upon the relaxation of red states, a ternary complex with the carotenoid is formed. Analysis of spectral signatures and interaction energies indicates that the specific iodo-tyrosine configuration enhances interactions between the carotenoid and W288.
Asunto(s)
Yodo , Triptófano , Aminoácidos , Enlace de Hidrógeno , Proteínas Bacterianas/metabolismo , Fluorescencia , Luz , Carotenoides/metabolismoRESUMEN
Our analysis of the X-ray crystal structure of canthaxanthin (CAN) showed that its ketolated ß-ionone rings can adopt two energetically equal, but structurally distinct puckers. Quantum chemistry calculations revealed that the potential energy surface of the ß-ionone ring rotation over the plane of the conjugated π-system in carotenoids depends on the pucker state of the ß-ring. Considering different pucker states and ß-ionone ring rotation, we found six separate local minima on the potential energy surface defining the geometry of the keto-ß-ionone ring-two cis and one trans orientation for each of two pucker states. We observed a small difference in energy and no difference in relative orientation for the cis-minima, but a pronounced difference for the position of trans-minimum in alternative pucker configurations. An energetic advantage of ß-ionone ring rotation from a specific pucker type can reach up to 8 kJ/mol ([Formula: see text]). In addition, we performed the simulation of linear absorption of CAN in hexane and in a unit cell of the CAN crystal. The electronic energies of [Formula: see text] transition were estimated both for the CAN monomer and in the CAN crystal. The difference between them reached [Formula: see text], which roughly corresponds to the energy gap between A and B pucker states predicted by theoretical estimations. Finally, we have discussed the importance of such effects for biological systems whose local environment determines conformational mobility, and optical/functional characteristics of carotenoid.
Asunto(s)
Carotenoides , Norisoprenoides , Carotenoides/química , Norisoprenoides/química , Conformación Molecular , CantaxantinaRESUMEN
Binding of dinitrosyl iron complex (DNIC) to albumin was studied using time-resolved fluorescence (TRF) and electron spin resonance (ESR) spectroscopy. It was found that the fluorescence lifetime of bovine serum albumin (BSA) and human serum albumin (HSA) decreases with binding and depends on DNIC concentration. The observed biexponential pattern of the BSA tryptophan (Trp) fluorescence decay is explained by the presence of two tryptophan residues in the protein molecule. We believe that DNIC forms stable complexes with the cysteine (Cys34) residue in the domain I of albumin. It was shown that the lifetime of albumin tryptophan fluorescence decreased during co-incubation of BSA with DNICs and glutathione. Effects of DNIC on the binding of specific spin-labeled fatty acids with albumin in human blood plasma were studied in vitro. The presence of DNIC in blood plasma does not change conformation of albumin domains II and III. We suggest that the most possible interaction between DNICs and albumin is the formation of a complex; and nitrosylation of the cysteine residue in the albumin domain I occurs without the changes in albumin conformation.
Asunto(s)
Hierro/farmacología , Óxidos de Nitrógeno/farmacología , Albúmina Sérica Bovina/efectos de los fármacos , Albúmina Sérica/efectos de los fármacos , Albúmina Sérica/metabolismo , Adulto , Anciano , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Glutatión/química , Humanos , Hierro/química , Masculino , Persona de Mediana Edad , Óxidos de Nitrógeno/química , Conformación Proteica , Albúmina Sérica/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de FluorescenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Colorantes Fluorescentes/química , Fotoperiodo , Modelos Moleculares , Conformación Proteica , Rodaminas/química , Solventes/química , Espectrometría de Fluorescencia , Temperatura , ViscosidadRESUMEN
In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ficobilisomas/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Carotenoides/metabolismo , Fluorescencia , Luz , Modelos Moleculares , Mutación/genética , Multimerización de Proteína/fisiologíaRESUMEN
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.
Asunto(s)
Respuesta al Choque por Frío/fisiología , Fluidez de la Membrana/fisiología , Synechocystis/fisiología , Anisotropía , Frío , Ácidos Grasos/metabolismo , Fluorescencia , Regulación Bacteriana de la Expresión Génica , Cinética , Lípidos de la Membrana/metabolismo , Oxidación-Reducción , Plastoquinona/metabolismo , Espectrometría Raman , Synechocystis/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Luminiscentes/química , Simulación de Dinámica Molecular , Absorción de Radiación , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cianobacterias/química , Colorantes Fluorescentes/farmacología , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP, as well as with the rotation of the echinenone's ß-ionone ring in the CTD. Structural data also helped rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.
Asunto(s)
Proteínas Bacterianas , Synechocystis , Proteínas Bacterianas/química , Synechocystis/metabolismo , Análisis Espectral , Carotenoides/química , Ficobilisomas/químicaRESUMEN
Phycobilisomes (PBSs) are giant water-soluble light-harvesting complexes of cyanobacteria and red algae, consisting of hundreds of phycobiliproteins precisely organized to deliver the energy of absorbed light to chlorophyll chromophores of the photosynthetic electron-transport chain. Quenching the excess of excitation energy is necessary for the photoprotection of photosynthetic apparatus. In cyanobacteria, quenching of PBS excitation is provided by the Orange Carotenoid Protein (OCP), which is activated under high light conditions. In this work, we describe parameters of anti-Stokes fluorescence of cyanobacterial PBSs in quenched and unquenched states. We compare the fluorescence readout from entire phycobilisomes and their fragments. The obtained results revealed the heterogeneity of conformations of chromophores in isolated phycobiliproteins, while such heterogeneity was not observed in the entire PBS. Under excitation by low-energy quanta, we did not detect a significant uphill energy transfer from the core to the peripheral rods of PBS, while the one from the terminal emitters to the bulk allophycocyanin chromophores is highly probable. We show that this direction of energy migration does not eliminate fluorescence quenching in the complex with OCP. Thus, long-wave excitation provides new insights into the pathways of energy conversion in the phycobilisome.
Asunto(s)
Cianobacterias , Ficobilisomas , Ficobilisomas/metabolismo , Proteínas Bacterianas/metabolismo , Fotosíntesis , Cianobacterias/metabolismo , Espectrometría de Fluorescencia/métodosRESUMEN
Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.
RESUMEN
Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.
Asunto(s)
Proteínas Bacterianas , Cianobacterias , Proteínas Bacterianas/química , Cianobacterias/metabolismo , Análisis Espectral , Transducción de Señal , Carotenoides/química , Ficobilisomas/químicaRESUMEN
The structural organization of natural pigment-protein complexes provides a specific environment for the chromophore groups. Yet, proteins are inherently dynamic and conformationally mobile. In this work, we demonstrate the heterogeneity of chromophores of C-phycocyanin (C-PC) from Arthrospira platensis. Part of the population of trimeric C-PC is subject to spontaneous disturbances of protein-protein interactions resulting in increased conformational mobility of the chromophores. Upon fluorescence excitation in the visible range, the spectral signatures of these poorly populated states are masked by bulk chromophore states, but the former could be clearly discriminated when the fluorescence is excited by near-infrared quanta. Such selective excitation of conformationally mobile C-PC chromophores is due to the structure of their S1 level, which is characterized by a significantly broadened spectral line. We demonstrate that the anti-Stokes C-PC fluorescence is the result of single-photon absorption. By combining spectral and structural methods, we characterize four distinct states of C-PC chromophores emitting at 620, 650, 665, and 720 nm and assigned the fast component in the anti-Stokes fluorescence decay kinetics in the range of 690-750 nm to the chromophores with increased conformational mobility. Our data suggest that the spectral and temporal characteristics of the anti-Stokes fluorescence can be used to study protein dynamics and develop methods to visualize local environment parameters such as temperature.
RESUMEN
The photoactive Orange Carotenoid Protein (OCP) plays a key role in cyanobacterial photoprotection. In OCP, a single non-covalently bound keto-carotenoid molecule acts as a light intensity sensor, while the protein is responsible for forming molecular contacts with the light-harvesting antenna, the fluorescence of which is quenched by OCP. Activation of this physiological interaction requires signal transduction from the photoexcited carotenoid to the protein matrix. Recent works revealed an asynchrony between conformational transitions of the carotenoid and the protein. Intrinsic tryptophan (Trp) fluorescence has provided valuable information about the protein part of OCP during its photocycle. However, wild-type OCP contains five Trp residues, which makes extraction of site-specific information impossible. In this work, we overcame this problem by characterizing the photocycle of a fully photoactive OCP variant (OCP-3FH) with only the most critical tryptophan residue (Trp-288) in place. Trp-288 is of special interest because it forms a hydrogen bond to the carotenoid's keto-oxygen to keep OCP in its dark-adapted state. Using femtosecond pump-probe fluorescence spectroscopy we analyzed the photocycle of OCP-3FH and determined the formation rate of the very first intermediate suggesting that generation of the recently discovered S* state of the carotenoid in OCP precedes the breakage of the hydrogen bonds. Therefore, following Trp fluorescence of the unique photoactive OCP-3FH variant, we identified the rate of the H-bond breakage and provided novel insights into early events accompanying photoactivation of wild-type OCP.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carotenoides/química , Triptófano/química , Proteínas Bacterianas/genética , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Espectrometría de FluorescenciaRESUMEN
The heterogeneity of metabolic reactions leads to a non-uniform distribution of temperature in different parts of the living cell. The demand to study normal functioning and pathological abnormalities of cellular processes requires the development of new visualization methods. Previously, we have shown that the 35-kDa photoswitchable Orange Carotenoid Protein (OCP) has a strong temperature dependency of photoconversion rates, and its tertiary structure undergoes significant structural rearrangements upon photoactivation, which makes this protein a nano-sized temperature sensor. However, the determination of OCP conversion rates requires measurements of carotenoid absorption, which is not suitable for microscopy. In order to solve this problem, we fused green and red fluorescent proteins (TagGFP and TagRFP) to the structure of OCP, producing photoactive chimeras. In such chimeras, electronic excitation of the fluorescent protein is effectively quenched by the carotenoid in OCP. Photoactivation of OCP-based chimeras triggers rearrangements of complex geometry, permitting measurements of the conversion rates by monitoring changes of fluorescence intensity. This approach allowed us to determine the local temperature of the microenvironment. Future directions to improve the OCP-based sensor are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carotenoides/metabolismo , Espectrometría de Fluorescencia/métodos , Temperatura , Transferencia Resonante de Energía de Fluorescencia , TermodinámicaRESUMEN
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.