RESUMO
Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.
Assuntos
Neoplasias da Mama , Transição Epitelial-Mesenquimal , Proteínas de Membrana , Fosfotransferases (Aceptor do Grupo Álcool) , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.
Assuntos
Complexos de Proteínas Captadores de Luz , Fotossíntese , Adaptação Fisiológica , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese/fisiologia , Plantas/metabolismo , Tilacoides/metabolismoRESUMO
When plants are exposed to high-light conditions, the potentially harmful excess energy is dissipated as heat, a process called non-photochemical quenching. Efficient energy dissipation can also be induced in the major light-harvesting complex of photosystem II (LHCII) in vitro, by altering the structure and interactions of several bound cofactors. In both cases, the extent of quenching has been correlated with conformational changes (twisting) affecting two bound carotenoids, neoxanthin, and one of the two luteins (in site L1). This lutein is directly involved in the quenching process, whereas neoxanthin senses the overall change in state without playing a direct role in energy dissipation. Here we describe the isolation of an intermediate state of LHCII, using the detergent n-dodecyl-α-D-maltoside, which exhibits the twisting of neoxanthin (along with changes in chlorophyll-protein interactions), in the absence of the L1 change or corresponding quenching. We demonstrate that neoxanthin is actually a reporter of the LHCII environment-probably reflecting a large-scale conformational change in the protein-whereas the appearance of excitation energy quenching is concomitant with the configuration change of the L1 carotenoid only, reflecting changes on a smaller scale. This unquenched LHCII intermediate, described here for the first time, provides for a deeper understanding of the molecular mechanism of quenching.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/químicaRESUMO
Carotenoids are conjugated linear molecules built from the repetition of terpene units, which display a large structural diversity in nature. They may, in particular, contain several types of side or end groups, which tune their functional properties, such as absorption position and photochemistry. We report here a detailed experimental study of the absorption and vibrational properties of allene-containing carotenoids, together with an extensive modeling of these experimental data. Our calculations can satisfactorily explain the electronic properties of vaucheriaxanthin, where the allene group introduces the equivalent of one CâC double bond into the conjugated CâC chain. The position of the electronic absorption of fucoxanthin and butanoyloxyfucoxanthin requires long-range corrections to be found correctly on the red side of that of vaucheriaxanthin; however, these corrections tend to overestimate the effect of the conjugated and nonconjugated CâO groups in these molecules. We show that the resonance Raman spectra of these carotenoids are largely perturbed by the presence of the allene group, with the two major Raman contributions split into two components. These perturbations are satisfactorily explained by modeling, through a gain in the Raman intensity of the CâC antisymmetric stretching mode, induced by the presence of the allene group in the carotenoid CâC chain.
Assuntos
Alcadienos , Carotenoides , Carotenoides/química , Eletrônica , Análise Espectral RamanRESUMO
Diatoms are a group of marine algae that are responsible for a significant part of global oxygen production. Adapted to life in an aqueous environment dominated by the blue-green light, their major light-harvesting antennae-fucoxanthin-chlorophyll protein complexes (FCPs)-exhibit different pigment compositions than of plants. Despite extensive experimental studies, until recently the theoretical description of excitation energy dynamics in these complexes was limited by the lack of high-resolution structural data. In this work, we use the recently resolved crystallographic information of the FCP complex from Phaeodactylum tricornutum diatom [Wang et al., Science 363, 6427 (2019)] and quantum chemistry-based calculations to evaluate the chlorophyll transition dipole moments, atomic transition charges from electrostatic potential, and the inter-chlorophyll couplings in this complex. The obtained structure-based excitonic couplings form the foundation for any modeling of stationary or time-resolved spectroscopic data. We also calculate the inter-pigment Förster energy transfer rates and identify two quickly equilibrating chlorophyll clusters.
Assuntos
Clorofila , Diatomáceas , Clorofila/química , Proteínas de Ligação à Clorofila/química , Proteínas de Ligação à Clorofila/metabolismo , Diatomáceas/química , Diatomáceas/metabolismo , Eletrônica , Complexos de Proteínas Captadores de Luz/química , Xantofilas/química , Xantofilas/metabolismoRESUMO
Diatoms are a major group of algae, responsible for a quarter of the global primary production on our planet. Their adaptation to marine environments is ensured by their light-harvesting antenna - the fucoxanthin-chlorophyll protein (FCP) complex, which absorbs strongly in the blue-green spectral region. Although these essential proteins have been the subject of many studies, for a long time their comprehensive description was not possible in the absence of structural data. Last year, the 3D structures of several FCP complexes were revealed. The structure of an FCP dimer was resolved by crystallography for the pennate diatom Phaeodactylum tricornutum [W. Wang et al., Science, 2019, 363, 6427] and the structure of the PSII supercomplex from the centric diatom Chaetoceros gracilis, containing several FCPs, was obtained by electron microscopy [X. Pi et al., Science, 2019, 365, 6452; R. Nagao et al., Nat. Plants, 2019, 5, 890]. In this Perspective article, we evaluate how precisely these structures may account for previously published ultrafast spectroscopy results, describing the excitation energy transfer in the FCP from another centric diatom Cyclotella meneghiniana. Surprisingly, we find that the published FCP structures cannot explain several observations obtained from ultrafast spectroscopy. Using the available structures, and results from electron microscopy, we construct a trimer-based FCP model for Cyclotella meneghiniana, consistent with ultrafast experimental data. As a whole, our observations suggest that the structures from the proteins belonging to the FCP family display larger variations than the equivalent LHC proteins in plants, which may reflect species-specific adaptations or original strategies for adapting to rapidly changing marine environments.
Assuntos
Clorofila A/química , Complexos de Proteínas Captadores de Luz/química , Xantofilas/química , Sequência de Aminoácidos , Clorofila/química , Diatomáceas/química , Transferência de Energia , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
We have investigated the photophysics of aggregated lutein/violaxanthin in daffodil chromoplasts. We reveal the presence of three carotenoid aggregate species, the main one composed of a mixture of lutein/violaxanthin absorbing at 481 nm, and two secondary populations of aggregated carotenoids absorbing circa 500 and 402 nm. The major population exhibits an efficient singlet fission process, generating µs-lived triplet states on an ultrafast timescale. The structural organization of aggregated lutein/violaxanthin in daffodil chromoplasts produces well-defined electronic levels that permit the energetic pathways to be disentangled unequivocally, allowing us to propose a consistent mechanism for singlet fission in carotenoid aggregates. Transient absorption measurements on this system reveal for the first time an entangled triplet signature for carotenoid aggregates, and its evolution into dissociated triplet states. A clear picture of the carotenoid singlet fission pathway is obtained, which is usually blurred due to the intrinsic disorder of carotenoid aggregates.
Assuntos
Corantes Fluorescentes/química , Luteína/química , Dimerização , Cinética , Conformação Molecular , Processos Fotoquímicos , Plastídeos/química , Espectrometria de Fluorescência , Xantofilas/químicaRESUMO
We report the functionalization of chalcogenide thin films with biotinylated 12-mer peptides SVSVGMKPSPRP and LLADTTHHRPWT exhibiting a high binding affinity toward inorganic surfaces, on the one hand, and with (3-aminopropyl)triethoxysilane (APTES), on the other hand. The specific biotin moieties were used to bind streptavidin proteins and demonstrate the efficacy of the biofunctionalizated chalcogenide thin films to capture biomolecules. Atomic force microscopy provided high-resolution images of the interfaces, and water contact angle measurements gave insight into the interaction mechanisms. Fourier transform infrared spectroscopy in attenuated total reflection mode provided information about the secondary structure of the bound proteins, thanks to the deconvolution of the amide I band (1700-1600 cm-1). Following adsorption of the biotinylated peptides or APTES immobilization, a homogenous coverage of the biotin layer exhibiting very low roughness was obtained, also rendering more hydrophilic Ge-Se-Te surfaces. Subsequent capture of streptavidin depends on the functionalization approach, permitting more or less an optimal orientation of the biotin to bind streptavidin. The molecular interface layer formed on Ge-Se-Te is crucial also for retaining the native secondary structure of the protein. Altogether, our results demonstrate that both peptides and APTES were appropriate linkers to build a favorable interface on chalcogenide materials to capture proteins, opening hereby promising biosensing applications.
RESUMO
Calculating the spectroscopic properties of complex conjugated organic molecules in their relaxed state is far from simple. An additional complexity arises for flexible molecules in solution, where the rotational energy barriers are low enough so that nonminimum conformations may become dynamically populated. These metastable conformations quickly relax during the minimization procedures preliminary to density functional theory calculations, and so accounting for their contribution to the experimentally observed properties is problematic. We describe a strategy for stabilizing these nonminimum conformations in silico, allowing their properties to be calculated. Diadinoxanthin and alloxanthin present atypical vibrational properties in solution, indicating the presence of several conformations. Performing energy calculations in vacuo and polarizable continuum model calculations in different solvents, we found three different conformations with values for the δ dihedral angle of the end ring ca. 0, 180, and 90° with respect to the plane of the conjugated chain. The latter conformation, a nonglobal minimum, is not stable during the minimization necessary for modeling its spectroscopic properties. To circumvent this classical problem, we used a Car-Parinello MD supermolecular approach, in which diadinoxanthin was solvated by water molecules so that metastable conformations were stabilized by hydrogen-bonding interactions. We progressively removed the number of solvating waters to find the minimum required for this stabilization. This strategy represents the first modeling of a carotenoid in a distorted conformation and provides an accurate interpretation of the experimental data.
RESUMO
In photosynthetic organisms, protection against photooxidative stress due to singlet oxygen is provided by carotenoid molecules, which quench chlorophyll triplet species before they can sensitize singlet oxygen formation. In anoxygenic photosynthetic organisms, in which exposure to oxygen is low, chlorophyll-to-carotenoid triplet-triplet energy transfer (T-TET) is slow, in the tens of nanoseconds range, whereas it is ultrafast in the oxygen-rich chloroplasts of oxygen-evolving photosynthetic organisms. To better understand the structural features and resulting electronic coupling that leads to T-TET dynamics adapted to ambient oxygen activity, we have carried out experimental and theoretical studies of two isomeric carotenoporphyrin molecular dyads having different conformations and therefore different interchromophore electronic interactions. This pair of dyads reproduces the characteristics of fast and slow T-TET, including a resonance Raman-based spectroscopic marker of strong electronic coupling and fast T-TET that has been observed in photosynthesis. As identified by density functional theory (DFT) calculations, the spectroscopic marker associated with fast T-TET is due primarily to a geometrical perturbation of the carotenoid backbone in the triplet state induced by the interchromophore interaction. This is also the case for the natural systems, as demonstrated by the hybrid quantum mechanics/molecular mechanics (QM/MM) simulations of light-harvesting proteins from oxygenic (LHCII) and anoxygenic organisms (LH2). Both DFT and electron paramagnetic resonance (EPR) analyses further indicate that, upon T-TET, the triplet wave function is localized on the carotenoid in both dyads.
Assuntos
Clorofila/química , Transferência de Energia , Fotossíntese , Carotenoides/química , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Complexos de Proteínas Captadores de Luz , Luteína/química , Modelos Moleculares , Conformação Molecular , Oxigênio , Pigmentação , Porfirinas/química , Teoria Quântica , Espectrofotometria , Análise Espectral RamanRESUMO
It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric 'ring' of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 'rings' are present in "low-light" adapted cells and that an unknown chaperon process creates multiple sub-types of 'rings' with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.
Assuntos
Apoproteínas/química , Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Rodopseudomonas/química , Espectrometria de FluorescênciaRESUMO
Cyanobacteria possess a family of one-helix high light-inducible proteins (Hlips) that are homologous to light-harvesting antenna of plants and algae. An Hlip protein, high light-inducible protein D (HliD) purified as a small complex with the Ycf39 protein is evaluated using resonance Raman spectroscopy. We show that the HliD binds two different ß-carotenes, each present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 489 and 522 nm, respectively. Both populations of ß-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted ß-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted ß-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its ß-rings. This second ß-carotene has highly twisted ß-rings adopting a flat conformation, which implies that the effective conjugation length N is extended up to 10.5 modifying the energetic levels. This increase in N will also result in a lower S1 energy state, which may provide a permanent energy dissipation channel. Analysis of the carbonyl stretching region for chlorophyll a excitations indicates that the HliD binds six chlorophyll a molecules in five non-equivalent binding sites, with at least one chlorophyll a presenting a slight distortion to its macrocycle. The binding modes and conformations of HliD-bound pigments are discussed with respect to the known structures of LHCII and CP29.
Assuntos
Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Synechocystis/química , beta Caroteno/química , Proteínas de Bactérias/genética , Complexos de Proteínas Captadores de Luz/genética , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Synechocystis/genética , beta Caroteno/genéticaRESUMO
Excitation energy transfer (EET) and trapping in Synechococcus WH 7803 whole cells and isolated photosystem I (PSI) complexes have been studied by time-resolved emission spectroscopy at room temperature (RT) and at 77 K. With the help of global and target analysis, the pathways of EET and the charge separation dynamics have been identified. Energy absorbed in the phycobilisome (PB) rods by the abundant phycoerythrin (PE) is funneled to phycocyanin (PC645) and from there to the core that contains allophycocyanin (APC660 and APC680). Intra-PB EET rates have been estimated to range from 11 to 68/ns. It was estimated that at RT, the terminal emitter of the phycobilisome, APC680, transfers its energy at a rate of 90/ns to PSI and at a rate of 50/ns to PSII. At 77 K, the redshifted Chl a states in the PSI core were heterogeneous, with maximum emission at 697 and 707 nm. In 72% of the PSI complexes, the bulk Chl a in equilibrium with F697 decayed with a main trapping lifetime of 39 ps.
Assuntos
Transferência de Energia , Synechococcus/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Especificidade da Espécie , Espectrometria de Fluorescência , Synechococcus/citologia , TemperaturaRESUMO
In the original publication, under the subtitle Recovery: fluorescence recovery protein (FRP), paragraph 4 the text section enclosed in quotation marks does not occur in one of the original publications cited (Sluchanko et al. 2017a, b).
RESUMO
Cyanobacteria exhibit a novel form of non-photochemical quenching (NPQ) at the level of the phycobilisome. NPQ is a process that protects photosystem II (PSII) from possible highlight-induced photo-damage. Although significant advancement has been made in understanding the NPQ, there are still some missing details. This critical review focuses on how the orange carotenoid protein (OCP) and its partner fluorescence recovery protein (FRP) control the extent of quenching. What is and what is not known about the NPQ is discussed under four subtitles; where does exactly the site of quenching lie? (site), how is the quenching being triggered? (trigger), molecular mechanism of quenching (quenching) and recovery from quenching. Finally, a recent working model of NPQ, consistent with recent findings, is been described.
Assuntos
Cianobactérias/fisiologia , Ficobilissomas/fisiologia , Regulação Bacteriana da Expressão Gênica , Processos Fotoquímicos , Complexo de Proteína do Fotossistema II/fisiologia , Conformação ProteicaRESUMO
The soil chromophyte alga Xanthonema (X.) debile contains only non-carbonyl carotenoids and Chl-a. X. debile has an antenna system denoted Xanthophyte light-harvesting complex (XLH) that contains the carotenoids diadinoxanthin, heteroxanthin, and vaucheriaxanthin. The XLH pigment stoichiometry was calculated by chromatographic techniques and the pigment-binding structure studied by resonance Raman spectroscopy. The pigment ratio obtained by HPLC was found to be close to 8:1:2:1 Chl-a:heteroxanthin:diadinoxanthin:vaucheriaxanthin. The resonance Raman spectra suggest the presence of 8-10 Chl-a, all of which are 5-coordinated to the central Mg, with 1-3 Chl-a possessing a macrocycle distorted from the relaxed conformation. The three populations of carotenoids are in the all-trans configuration. Vaucheriaxanthin absorbs around 500-530 nm, diadinoxanthin at 494 nm and heteroxanthin at 487 nm at 4.5 K. The effective conjugation length of heteroxanthin and diadinoxanthin has been determined as 9.4 in both cases; the environment polarizability of the heteroxanthin and diadinoxanthin binding pockets is 0.270 and 0.305, respectively.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Estramenópilas/química , Carotenoides/química , Cromatografia Líquida de Alta Pressão , Conformação Proteica , Análise Espectral RamanRESUMO
Cyanobacteria perform photosynthesis with the use of large light-harvesting antennae called phycobilisomes (PBSs). These hemispherical PBSs contain hundreds of open-chain tetrapyrrole chromophores bound to different peptides, providing an arrangement in which excitation energy is funnelled towards the PBS core from where it can be transferred to photosystem I and/or photosystem II. In the PBS core, many allophycocyanin (APC) trimers are present, red-light-absorbing phycobiliproteins that covalently bind phycocyanobilin (PCB) chromophores. APC trimers were amongst the first light-harvesting complexes to be crystallized. APC trimers have two spectrally different PCBs per monomer, a high- and a low-energy pigment. The crystal structure of the APC trimer reveals the close distance (~21 Å) between those two chromophores (the distance within one monomer is ~51 Å) and this explains the ultrafast (~1 ps) excitation energy transfer (EET) between them. Both chromophores adopt a somewhat different structure, which is held responsible for their spectral difference. Here we used spectrally resolved picosecond fluorescence to study EET in these APC trimers both in crystallized and in solubilized form. We found that not all closely spaced pigment couples consist of a low- and a high-energy pigment. In ~10% of the cases, a couple consists of two high-energy pigments. EET to a low-energy pigment, which can spectrally be resolved, occurs on a time scale of tens of picoseconds. This transfer turns out to be three times faster in the crystal than in the solution. The spectral characteristics and the time scale of this transfer component are similar to what have been observed in the whole cells of Synechocystis sp. PCC 6803, for which it was ascribed to EET from C-phycocyanin to APC. The present results thus demonstrate that part of this transfer should probably also be ascribed to EET within APC trimers.
Assuntos
Transferência de Energia , Ficocianina/química , Cristalografia por Raios X , Microscopia de Fluorescência , Conformação Molecular , Soluções , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and ß-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and ß-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound ß-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two ß-carotenes.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clorofila/metabolismo , Synechocystis/metabolismo , beta Caroteno/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise Espectral Raman , Synechocystis/genética , Synechocystis/fisiologiaRESUMO
Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Análise Espectral Raman/métodos , Sítios de Ligação , Clorofila/química , Clorofila A , Luteína/química , Mutação , Xantofilas/químicaRESUMO
Resonance Raman spectroscopy was used to evaluate pigment structure in the FCP-like light-harvesting complex of Chromera velia (Chromera light-harvesting complex or CLH). This antenna protein contains chlorophyll a, violaxanthin and a new isofucoxanthin-like carotenoid (called Ifx-l). We show that Ifx-l is present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 515 and 548nm respectively. In this complex, only one violaxanthin population absorbing at 486nm is observed. All the CLH-bound carotenoid molecules are in all-trans configuration, and among the two Ifx-l carotenoid molecules, the red one is twisted, as is the red-absorbing lutein in LHCII trimers. Analysis of the carbonyl stretching region for Chl a excitations indicates CLH binds up to seven Chl a molecules in five non-equivalent binding sites, in reasonable agreement with sequence analyses which have identified eight potential coordinating residues. The binding modes and conformations of CLH-bound pigments are discussed with respect to the known structures of LHCII and FCP.