Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

• Klíčová slova
• Chromera velia, antenna proteins, fluorescence correlation spectroscopy, light-harvesting, microscopy, photosynthesis, protein diffusion, protein oligomerization,
• MeSH
• bílkoviny řas chemie   metabolismus   MeSH
• fluorescence * MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza * MeSH
• kinetika MeSH
• multimerizace proteinu MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

• MeSH
• Arabidopsis chemie   ultrastruktura   MeSH
• Chlamydomonas reinhardtii chemie   ultrastruktura   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• podjednotky proteinů MeSH
• světlosběrné proteinové komplexy chemie   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• podjednotky proteinů MeSH
• světlosběrné proteinové komplexy MeSH

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.

• Klíčová slova
• Algae, Carotenoids, Chl-a, Diadinoxanthin, Heteroxanthin, Light-harvesting complex, Resonance Raman,
• MeSH
• Heterokontophyta chemie   MeSH
• karotenoidy chemie   MeSH
• konformace proteinů MeSH
• Ramanova spektroskopie MeSH
• světlosběrné proteinové komplexy chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karotenoidy MeSH
• světlosběrné proteinové komplexy MeSH

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 β-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 β-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 β-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 β-carotenes.

• Klíčová slova
• Chlorophyll, HLIPs, HliC, Raman spectroscopy, Synechocystis, β-Carotene,
• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• beta-karoten metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• multimerizace proteinu MeSH
• Ramanova spektroskopie MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• světlosběrné proteinové komplexy genetika   metabolismus   MeSH
• Synechocystis genetika   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-karoten MeSH
• chlorofyl MeSH
• high light-inducible protein, cyanobacteria MeSH Prohlížeč
• rekombinantní proteiny MeSH
• světlosběrné proteinové komplexy MeSH

In oxygenic photosynthesis the initial photochemical processes are carried out by photosystem I (PSI) and II (PSII). Although subunit composition varies between cyanobacterial and plastid photosystems, the core structures of PSI and PSII are conserved throughout photosynthetic eukaryotes. So far, the photosynthetic complexes have been characterised in only a small number of organisms. We performed in silico and biochemical studies to explore the organization and evolution of the photosynthetic apparatus in the chromerids Chromera velia and Vitrella brassicaformis, autotrophic relatives of apicomplexans. We catalogued the presence and location of genes coding for conserved subunits of the photosystems as well as cytochrome b6f and ATP synthase in chromerids and other phototrophs and performed a phylogenetic analysis. We then characterised the photosynthetic complexes of Chromera and Vitrella using 2D gels combined with mass-spectrometry and further analysed the purified Chromera PSI. Our data suggest that the photosynthetic apparatus of chromerids underwent unique structural changes. Both photosystems (as well as cytochrome b6f and ATP synthase) lost several canonical subunits, while PSI gained one superoxide dismutase (Vitrella) or two superoxide dismutases and several unknown proteins (Chromera) as new regular subunits. We discuss these results in light of the extraordinarily efficient photosynthetic processes described in Chromera.

• MeSH
• Alveolata genetika   fyziologie   MeSH
• delece genu MeSH
• fotosyntéza genetika   fyziologie   MeSH
• fotosystém I (proteinový komplex) genetika   izolace a purifikace   fyziologie   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie MeSH
• molekulární evoluce MeSH
• superoxiddismutasa metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém I (proteinový komplex) MeSH
• superoxiddismutasa MeSH

Resonance Raman spectroscopy was used to evaluate pigment-binding site properties in the violaxanthin-chlorophyll-a-binding protein (VCP) from Nannochloropsis oceanica. The pigments bound to this antenna protein are chlorophyll-a, violaxanthin, and vaucheriaxanthin. The molecular structures of bound Chl-a molecules are discussed with respect to those of the plant antenna proteins LHCII and CP29, the crystal structures of which are known. We show that three populations of carotenoid molecules are bound by VCP, each of which is in an all-trans configuration. We assign the lower-energy absorption transition of each of these as follows. One violaxanthin population absorbs at 485 nm, while the second population is red-shifted and absorbs at 503 nm. The vaucheriaxanthin population absorbs at 525 nm, a position red-shifted by 2138 cm-1 as compared to isolated vaucheriaxanthin in n-hexane. The red-shifted violaxanthin is slightly less planar than the blue-absorbing one, as observed for the two central luteins in LHCII, and we suggest that these violaxanthins occupy the two equivalent binding sites in VCP at the centre of the cross-brace. The presence of a highly red-shifted vaucheriaxanthin in VCP is reminiscent of the situation of FCP, in which (even more) highly red-shifted populations of fucoxanthin are present. Tuning carotenoids to absorb in the green-yellow region of the visible spectrum appears to be a common evolutionary response to competition with other photosynthetic species in the aquatic environment.

• Klíčová slova
• Carotenoids, Light-harvesting complex, Nannochloropsis oceanica, Resonance Raman, VCP,
• MeSH
• chlorofyl chemie   MeSH
• karotenoidy chemie   MeSH
• Ramanova spektroskopie MeSH
• světlosběrné proteinové komplexy chemie   MeSH
• transportní proteiny chemie   MeSH
• xanthofyly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• karotenoidy MeSH
• světlosběrné proteinové komplexy MeSH
• transportní proteiny MeSH
• violaxanthin MeSH Prohlížeč
• xanthofyly MeSH

Photosystem I (PSI) is a multi-subunit integral pigment-protein complex that performs light-driven electron transfer from plastocyanin to ferredoxin in the thylakoid membrane of oxygenic photoautotrophs. In order to achieve the optimal photosynthetic performance under ambient irradiance, the absorption cross section of PSI is extended by means of peripheral antenna complexes. In eukaryotes, this role is played mostly by the pigment-protein complexes of the LHC family. The structure of the PSI-antenna supercomplexes has been relatively well understood in organisms harboring the primary plastid: red algae, green algae and plants. The secondary endosymbiotic algae, despite their major ecological importance, have so far received less attention. Here we report a detailed structural analysis of the antenna-PSI association in the stramenopile alga Nannochloropsis oceanica (Eustigmatophyceae). Several types of PSI-antenna assemblies are identified allowing for identification of antenna docking sites on the PSI core. Instances of departure of the stramenopile system from the red algal model of PSI-Lhcr structure are recorded, and evolutionary implications of these observations are discussed.

• Klíčová slova
• Electron microscopy, Light-harvesting complex, Nannochloropsis, Photosystem I, Stramenopila,
• MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• plastidy metabolismus   MeSH
• Rhodophyta metabolismus   MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fotosystém I (proteinový komplex) MeSH

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 β-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 β-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted β-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted β-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its β-rings. This second β-carotene has highly twisted β-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.

• Klíčová slova
• carotenoid, chlorophyll, cyanobacteria, light-harvesting complex (antenna complex), photosynthesis,
• MeSH
• bakteriální proteiny chemie   genetika   MeSH
• beta-karoten chemie   genetika   MeSH
• kvarterní struktura proteinů MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• světlosběrné proteinové komplexy chemie   genetika   MeSH
• Synechocystis chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-karoten MeSH
• světlosběrné proteinové komplexy MeSH

Plants collect light for photosynthesis using light-harvesting complexes (LHCs)-an array of chlorophyll proteins that are able to reversibly switch from harvesting to energy-dissipation mode to prevent damage of the photosynthetic apparatus. LHC antennae as well as other members of the LHC superfamily evolved from cyanobacterial ancestors called high light-inducible proteins (Hlips). Here, we characterized a purified Hlip family member HliD isolated from the cyanobacterium Synechocystis sp. PCC 6803. We found that the HliD binds chlorophyll-a (Chl-a) and β-carotene and exhibits an energy-dissipative conformation. Using femtosecond spectroscopy, we demonstrated that the energy dissipation is achieved via direct energy transfer from a Chl-a Qy state to the β-carotene S1 state. We did not detect any cation of β-carotene that would accompany Chl-a quenching. These results provide proof of principle that this quenching mechanism operates in the LHC superfamily and also shed light on the photoprotective role of Hlips and the evolution of LHC antennae.

• MeSH
• beta-karoten chemie   MeSH
• chlorofyl a MeSH
• chlorofyl chemie   MeSH
• elektrony MeSH
• fluorescenční spektrometrie MeSH
• fotochemické procesy * MeSH
• fotosyntéza MeSH
• karotenoidy chemie   MeSH
• konformace proteinů MeSH
• přenos energie MeSH
• rostlinné proteiny chemie   MeSH
• rostliny metabolismus   MeSH
• sinice metabolismus   MeSH
• spektrofotometrie MeSH
• světlo MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Synechocystis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-carotene MeSH Prohlížeč
• beta-karoten MeSH
• chlorofyl a MeSH
• chlorofyl MeSH
• karotenoidy MeSH
• rostlinné proteiny MeSH
• světlosběrné proteinové komplexy MeSH

Red algae represent an evolutionarily important group that gave rise to the whole red clade of photosynthetic organisms. They contain a unique combination of light-harvesting systems represented by a membrane-bound antenna and by phycobilisomes situated on thylakoid membrane surfaces. So far, very little has been revealed about the mobility of their phycobilisomes and the regulation of their light-harvesting system in general. Therefore, we carried out a detailed analysis of phycobilisome dynamics in several red alga strains and compared these results with the presence (or absence) of photoprotective mechanisms. Our data conclusively prove phycobilisome mobility in two model mesophilic red alga strains, Porphyridium cruentum and Rhodella violacea. In contrast, there was almost no phycobilisome mobility in the thermophilic red alga Cyanidium caldarium that was not caused by a decrease in lipid desaturation in this extremophile. Experimental data attributed this immobility to the strong phycobilisome-photosystem interaction that highly restricted phycobilisome movement. Variations in phycobilisome mobility reflect the different ways in which light-harvesting antennae can be regulated in mesophilic and thermophilic red algae. Fluorescence changes attributed in cyanobacteria to state transitions were observed only in mesophilic P. cruentum with mobile phycobilisomes, and they were absent in the extremophilic C. caldarium with immobile phycobilisomes. We suggest that state transitions have an important regulatory function in mesophilic red algae; however, in thermophilic red algae, this process is replaced by nonphotochemical quenching.

• Publikační typ
• časopisecké články MeSH