RESUMO
The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5â Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αß-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex.
Assuntos
Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Complexos de Proteínas Captadores de Luz/química , Rhodospirillum rubrum/química , Proteínas de Bactérias/isolamento & purificação , Bacterioclorofilas/química , Benzoquinonas/química , Sítios de Ligação , Cristalização , Complexo III da Cadeia de Transporte de Elétrons/química , Ligação de Hidrogênio , Hidroquinonas/química , Ligantes , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Fosfolipídeos/química , Conformação Proteica em alfa-HéliceRESUMO
Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI-PSII megacomplex, PSII-LHCII, two PSI-LHCI-LHCIIs, PSI-LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI-LHCI-LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII-LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI-LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.
Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Estreptófitas/metabolismo , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Espectrometria de Massas , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/isolamento & purificação , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Filogenia , Análise de Sequência de DNA , Estreptófitas/genéticaRESUMO
Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.
Assuntos
Arabidopsis/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Tilacoides/metabolismo , Arabidopsis/efeitos dos fármacos , Cinética , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Lincomicina/farmacologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Multimerização Proteica , Espectrometria de Fluorescência , Ultracentrifugação , Xantofilas/química , Xantofilas/metabolismo , Zeaxantinas/química , Zeaxantinas/metabolismoRESUMO
Light-harvesting complex II (LHCII) absorbs light energy and transfers it primarily to photosystem II in green algae and land plants. Although the trimeric structure of LHCII is conserved between the two lineages, its subunit composition and function are believed to differ significantly. In this study, we purified four LHCII trimers from the green alga Chlamydomonas reinhardtii and analyzed their biochemical properties. We used several preparation methods to obtain four distinct fractions (fractions 1-4), each of which contained an LHCII trimer with different contents of Type I, III, and IV proteins. The pigment compositions of the LHCIIs in the four fractions were similar. The absorption and fluorescence spectra were also similar, although the peak positions differed slightly. These results indicate that this green alga contains four types of LHCII trimer with different biochemical and spectroscopic features. Based on these findings, we discuss the function and structural organization of green algal LHCII antennae.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Multimerização Proteica , Carotenoides/metabolismo , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Subunidades Proteicas/metabolismo , Espectrometria de Fluorescência , TemperaturaRESUMO
Photosystem I (PSI) is a highly efficient natural light-energy converter, and has diverse light-harvesting antennas associated with its core in different photosynthetic organisms. In green algae, an extremely large light-harvesting complex I (LHCI) captures and transfers energy to the PSI core. Here, we report the structure of PSI-LHCI from a green alga Bryopsis corticulans at 3.49 Å resolution, obtained by single-particle cryo-electron microscopy, which revealed 13 core subunits including subunits characteristic of both prokaryotes and eukaryotes, and 10 light-harvesting complex a (Lhca) antennas that form a double semi-ring and an additional Lhca dimer, including a novel four-transmembrane-helix Lhca. In total, 244 chlorophylls were identified, some of which were located at key positions for the fast energy transfer. These results provide a firm structural basis for unravelling the mechanisms of light-energy harvesting, transfer and quenching in the green algal PSI-LHCI, and important clues as to how PSI-LHCI has changed during evolution.
Assuntos
Clorófitas/química , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Clorofila/química , Clorofila/metabolismo , Clorófitas/genética , Microscopia Crioeletrônica , Transferência de Energia , Evolução Molecular , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/isolamento & purificação , Complexo de Proteína do Fotossistema I/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
The colonial green alga Botryococcus braunii (BB) is a potential source of biofuel due to its natural high hydrocarbon content. Unfortunately, its slow growth limits its biotechnological potential. Understanding its photosynthetic machinery could help to identify possible growth limitations. Here, we present the first study on BB light-harvesting complexes (LHCs). We purified two LHC fractions containing the complexes in monomeric and trimeric form. Both fractions contained at least two proteins with molecular weight (MW) around 25 kDa. The chlorophyll composition is similar to that of the LHCII of plants; in contrast, the main xanthophyll is loroxanthin, which substitutes lutein in most binding sites. Circular dichroism and 77 K absorption spectra lack typical differences between monomeric and trimeric complexes, suggesting that intermonomer interactions do not play a role in BB LHCs. This is in agreement with the low stability of the BB LHCII trimers as compared to the complexes of plants, which could be related to loroxanthin binding in the central (L1 and L2) binding sites. The properties of BB LHCII are similar to those of plant LHCII, indicating a similar pigment organization. Differences are a higher content of red chlorophyll a, similar to plant Lhcb3. These differences and the different Xan composition had no effect on excitation energy transfer or fluorescence lifetimes, which were similar to plant LHCII.
Assuntos
Clorófitas/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Plantas/metabolismo , Dicroísmo Circular , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Pigmentos Biológicos/metabolismo , Proteínas de Plantas/isolamento & purificação , Desnaturação Proteica , Estabilidade Proteica , Espectrometria de Fluorescência , Temperatura , Tilacoides/metabolismo , Fatores de TempoRESUMO
Light-harvesting complex 2 (LH2) is an integral membrane protein in purple photosynthetic bacteria. This protein possesses two types of bacteriochlorophyll (BChl) a, termed B800 and B850, which exhibit lowest-energy absorption bands (Qy bands) around 800 and 850 nm. These BChl a pigments in the LH2 protein play crucial roles not only in photosynthetic functions but also in folding and maintaining its protein structure. We report herein the reversible structural changes in the LH2 protein derived from a purple photosynthetic bacterium, Rhodoblastus acidophilus, induced by the removal of B800 BChl a (denoted as B800-free LH2) and the reconstitution of exogenous BChl a. Atomic force microscopy observation clearly visualized the nonameric ring structure of the B800-free LH2 with almost the same diameter as the native LH2. Size exclusion chromatography measurements indicated a considerable decrease in the size of the protein induced by the removal of B800 BChl a. The protein size was almost recovered by the insertion of BChl a pigments into the B800 binding sites. The decrease in the LH2 size would mainly originate from the shrinkage of the B800 binding sites perpendicular to the macrocycle of B800 BChl a without deformation of the circular arrangement. The reversible changes in the LH2 structure induced by the removal and reconstitution of B800 BChl a will be helpful for understanding the structural principle and the folding mechanism of photosynthetic pigment-protein complexes.
Assuntos
Proteínas de Bactérias/metabolismo , Bacterioclorofila A/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Pigmentos Biológicos/metabolismo , Rhodobacter sphaeroides/metabolismo , Rodopseudomonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Bacterioclorofila A/química , Bacterioclorofila A/isolamento & purificação , Sítios de Ligação , Cromatografia em Gel , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Microscopia de Força Atômica , Peso Molecular , Pigmentos Biológicos/química , Pigmentos Biológicos/isolamento & purificação , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Espectrofotometria UltravioletaRESUMO
This work discusses the protein conformational complexity of the B800-850 LH2 complexes from the purple sulfur bacterium Allochromatium vinosum, focusing on the spectral characteristics of the B850 chromophores. Low-temperature B850 absorption and the split B800 band shift blue and red, respectively, at elevated temperatures, revealing isosbestic points. The latter indicates the presence of two (unresolved) conformations of B850 bacteriochlorophylls (BChls), referred to as conformations 1 and 2, and two conformations of B800 BChls, denoted as B800R and B800B. The energy differences between average site energies of conformations 1 and 2, and B800R and B800B are similar (â¼200 cm-1), suggesting weak and strong hydrogen bonds linking two major subpopulations of BChls and the protein scaffolding. Although conformations 1 and 2 of the B850 chromophores, and B800R and B800B, exist in the ground state, selective excitation leads to 1 â 2 and B800R â B800B phototransformations. Different static inhomogeneous broadening is revealed for the lowest energy exciton states of B850 (fwhm â¼195 cm-1) and B800R (fwhm â¼140 cm-1). To describe the 5 K absorption spectrum and the above-mentioned conformations, we employ an exciton model with dichotomous protein conformation disorder. We show that both experimental data and the modeling study support a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls, which under illumination undergo conformational changes, most likely caused by proton dynamics.
Assuntos
Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Proteínas de Bactérias/isolamento & purificação , Chromatiaceae , Fluorescência , Ligação de Hidrogênio , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Modelos Químicos , Fotodegradação , Conformação Proteica , TemperaturaRESUMO
Photosystem I (PSI)-light-harvesting complex I (LHCI) super-complex and its sub-complexes PSI core and LHCI, were purified from a unicellular red alga Cyanidioschyzon merolae and characterized. PSI-LHCI of C. merolae existed as a monomer with a molecular mass of 580 kDa. Mass spectrometry analysis identified 11 subunits (PsaA, B, C, D, E, F, I, J, K, L, O) in the core complex and three LHCI subunits, CMQ142C, CMN234C, and CMN235C in LHCI, indicating that at least three Lhcr subunits associate with the red algal PSI core. PsaG was not found in the red algae PSI-LHCI, and we suggest that the position corresponding to Lhca1 in higher plant PSI-LHCI is empty in the red algal PSI-LHCI. The PSI-LHCI complex was separated into two bands on native PAGE, suggesting that two different complexes may be present with slightly different protein compositions probably with respective to the numbers of Lhcr subunits. Based on the results obtained, a structural model was proposed for the red algal PSI-LHCI. Furthermore, pigment analysis revealed that the C. merolae PSI-LHCI contained a large amount of zeaxanthin, which is mainly associated with the LHCI complex whereas little zeaxanthin was found in the PSI core. This indicates a unique feature of the carotenoid composition of the Lhcr proteins and may suggest an important role of Zea in the light-harvesting and photoprotection of the red algal PSI-LHCI complex.
Assuntos
Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexo de Proteína do Fotossistema I/isolamento & purificação , Rodófitas/metabolismo , Sequência de Aminoácidos , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Biológicos , Oxigênio/metabolismo , Peptídeos/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Pigmentos Biológicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Espectrometria de FluorescênciaRESUMO
Low molecular weight (MW) polyols are organic osmolytes influencing protein structure and activity. We have intended to investigate the effects of low MW polyols on the optical and the excited-state properties of the light-harvesting complex 2 (LH2) isolated from the photosynthetic bacterium Thermochromatium (Tch.) tepidum, a thermophile growing at â¼50 °C. Steady state spectroscopy demonstrated that, on increasing glycerol or sorbitol fractions up to 60% (polyol/water, v/v), the visible absorption of carotenoids (Crts) remained unchanged, while the near infrared Qy absorption of bacteriochlorophyll a (BChl) at 800 nm (B800) and 850 nm (B850) varied slightly. Further increasing the fraction of glycerol (but not sorbitol) to 80% (v/v) induced distinct changes of the near infrared absorption and fluorescence spectra. Transient absorption spectroscopy revealed that, following the fast processes of BChl-to-Crt triplet energy transfer, rather weak Qy signals of B800 and B850 remained and evolved in phase with the kinetics of triplet excited state Crt (3Crt*), which are attributed to the Qy band shift as a result of 3Crt*-BChl interaction. The steady state and the transient spectral responses of the Qy bands are found to correlate intimately with the water activity varying against polyol MW and mixing ratio, which are rationalized by the change of the hydration status of the C- and N-termini of LH2. Our results suggest that, with reference to the mesophilic purple bacterium Rhodobacter sphaeroides 2.4.1, Tch. tepidum adopts substantially more robust LH2 hydration against the osmotic effects from the low MW polyols.
Assuntos
Chromatiaceae/química , Complexos de Proteínas Captadores de Luz/química , Polímeros/química , Chromatiaceae/metabolismo , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Polímeros/metabolismo , Solventes/química , Solventes/metabolismo , Espectrometria de Fluorescência , Análise Espectral Raman , Água/química , Água/metabolismoRESUMO
Fucoxanthin is a carotenoid that is mainly found in light-harvesting complexes from brown algae and diatoms. Due to the presence of a carbonyl group attached to polyene chains in polar environments, excitation produces an excited intra-molecular charge transfer. This intra-molecular charge transfer state plays a key role in the highly efficient (â¼95%) energy-transfer from fucoxanthin to chlorophyll a in the light-harvesting complexes from brown algae. In purple bacterial light-harvesting systems the efficiency of excitation energy-transfer from carotenoids to bacteriochlorophylls depends on the extent of conjugation of the carotenoids. In this study we were successful, for the first time, in incorporating fucoxanthin into a light-harvesting complex 1 from the purple photosynthetic bacterium, Rhodospirillum rubrum G9+ (a carotenoidless strain). Femtosecond pump-probe spectroscopy was applied to this reconstituted light-harvesting complex in order to determine the efficiency of excitation energy-transfer from fucoxanthin to bacteriochlorophyll a when they are bound to the light-harvesting 1 apo-proteins.
Assuntos
Transferência de Energia , Complexos de Proteínas Captadores de Luz/metabolismo , Xantofilas/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Modelos Moleculares , Conformação Molecular , Rhodospirillum rubrum/enzimologia , Xantofilas/químicaRESUMO
This work focuses on the development of a molecular tool for purification of Photosystem II (PSII) from Nicotiana tabacum (L.). To this end, the chloroplast psbB gene encoding the CP47 PSII subunit was replaced with an engineered version of the same gene containing a C-terminal His-tag. Molecular analyses assessed the effective integration of the recombinant gene and its expression. Despite not exhibiting any obvious phenotype, the transplastomic plants remained heteroplasmic even after three rounds of regeneration under antibiotic selection. However, the recombinant His-tagged CP47 protein associated in vivo to the other PSII subunits allowing the isolation of a functional PSII core complex, although with low yield of extraction. These results will open up possible perspectives for further spectroscopic and structural studies.
Assuntos
Engenharia Genética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Nicotiana/genética , Nicotiana/metabolismo , Complexo de Proteína do Fotossistema II/isolamento & purificação , Plastídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Genes de Plantas , Vetores Genéticos/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação/genética , Fenótipo , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas , Subunidades Proteicas/metabolismo , Análise EspectralRESUMO
In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII) collect excitations and funnel these to the photosynthetic reaction center where charge separation takes place. In excess light conditions, remodeling of the photosynthetic membrane and protein conformational changes produces a photoprotective state in which excitations are rapidly quenched to avoid photodamage. The quenched states are associated with protein aggregation, however the LHCII complexes are also proposed to have an intrinsic capacity to shift between light harvesting and fluorescence-quenched conformational states. To disentangle the effects of protein-protein and protein-lipid interactions on the LHCII photoprotective switch, we compared the structural and fluorescent properties of LHCII lipid nanodiscs and proteoliposomes with very low protein-to-lipid ratios. We demonstrate that LHCII proteins adapta fully fluorescent state in nanodiscs and in proteoliposomes with highly diluted protein densities. Increasing the protein density induces a transition into a mildly-quenched state that reaches a plateau at a molar protein-to-lipid ratio of 0.001 and has a fluorescence yield reminiscent of the light-harvesting state in vivo. The low onset for quenching strongly suggests that LHCII-LHCII attractive interactions occur inside membranes. The transition at low protein densities does not involve strong changes in the excitonic circular-dichroism spectrum and is distinct from a transition occurring at very high protein densities that comprises strong fluorescence quenching and circular-dichroism spectral changes involving chlorophyll 611 and 612, correlating with proposed quencher sites of the photoprotective mechanisms.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Fosfatidilcolinas/química , Folhas de Planta/química , Proteolipídeos/química , Galactolipídeos/química , Galactolipídeos/metabolismo , Glicolipídeos/química , Glicolipídeos/metabolismo , Cinética , Luz , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Nanoestruturas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Folhas de Planta/fisiologia , Proteolipídeos/metabolismo , Espectrometria de Fluorescência , Spinacia oleracea/química , Spinacia oleracea/fisiologiaRESUMO
The light-harvesting 1 reaction center (LH1-RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermostability and an unusual LH1 Qy transition, both induced by Ca2+ binding. In this study, metal-binding sites and metal-protein interactions in the LH1-RC complexes from wild-type (B915) and biosynthetically Sr2+-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca2+, Sr2+, or Ba2+ to the LH1 αß-subunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca2+ and Sr2+ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca2+ and Sr2+ (or Ba2+) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca2+ depletion and Sr2+ substitution demonstrated that dissociation and binding of Ca2+ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr2+ (or Ba2+) exists in the vicinity of the Ca2+-binding site, a part of which is shared in both metal-binding sites.
Assuntos
Bário/química , Cálcio/química , Chromatiaceae/química , Complexos de Proteínas Captadores de Luz/química , Subunidades Proteicas/química , Estrôncio/química , Motivos de Aminoácidos , Anaerobiose/fisiologia , Bário/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , Chromatiaceae/fisiologia , Temperatura Alta , Cinética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Fotossíntese/fisiologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Estrôncio/metabolismo , TermodinâmicaRESUMO
Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation-bacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.
Assuntos
Carotenoides/química , Complexos de Proteínas Captadores de Luz/química , Teoria Quântica , Rhodobacter sphaeroides/química , Carotenoides/metabolismo , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Rhodobacter sphaeroides/crescimento & desenvolvimento , Rhodobacter sphaeroides/metabolismoRESUMO
The efficiency of visible light in inducing fluorescence quenching in the isolated light-harvesting complex II (LHCII) of higher plants is investigated by action spectroscopy in the visible portion of photosynthetic active radiation. The efficiency spectrum displays a relatively homogenous quenching yield across the most intense electronic transitions of the chlorophyll a and carotenoid pigments, indicating that quenching proceeds from the equilibrated state of the complex. Larger yields are observed in the 510-640-nm window, where weak transitions of LHCII-bound chromophores occur. This observation is interpreted in terms of an additional quenching sensitisation process mediated by these electronic transitions.
Assuntos
Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema II/genética , Spinacia oleracea/metabolismo , Carotenoides/biossíntese , Carotenoides/genética , Clorofila/genética , Clorofila A , Transporte de Elétrons/genética , Luz , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Fotossíntese/genética , Spinacia oleracea/genética , Tilacoides/metabolismoRESUMO
Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans-cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Luz , Proteínas de Plantas/química , Xantofilas/química , Isomerismo , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Microscopia Confocal , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Estrutura Quaternária de Proteína , Análise Espectral Raman , Spinacia oleracea/metabolismo , Xantofilas/isolamento & purificação , Xantofilas/metabolismoRESUMO
Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the ß2 adrenergic receptor (ß2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the ß2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.
Assuntos
Manitol/química , Proteínas de Membrana/química , Tensoativos/química , Antiporters/química , Antiporters/isolamento & purificação , Arabidopsis/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Estabilidade Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/isolamento & purificação , Rhodobacter capsulatus/química , SolubilidadeRESUMO
Non-photochemical quenching (NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ.
Assuntos
Alveolados/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Processos Fotoquímicos , Alveolados/citologia , Alveolados/efeitos da radiação , Clorofila/metabolismo , Fluorescência , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Fatores de Tempo , Xantofilas/metabolismoRESUMO
In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.