Observation of conformational dynamics in single light-harvesting proteins from cryptophyte algae.

Photosynthetic organisms use pigment-protein complexes to capture the sunlight that powers most life on earth. Within these complexes, the position of the embedded pigments is all optimized for light harvesting. At the same time, the protein scaffold undergoes thermal fluctuations that vary the structure, and, thus, photophysics, of the complexes. While these variations are averaged out in ensemble measurements, single-molecule spectroscopy provides the ability to probe these conformational changes. We used single-molecule fluorescence spectroscopy to identify the photophysical substates reflective of distinct conformations and the associated conformational dynamics in phycoerythrin 545 (PE545), a pigment-protein complex from cryptophyte algae. Rapid switching between photophysical states was observed, indicating that ensemble measurements average over a conformational equilibrium. A highly quenched conformation was also identified, and its population increased under high light. This discovery establishes that PE545 has the characteristics to serve as a photoprotective site. Finally, unlike homologous proteins from the evolutionarily related cyanobacteria and red algae, quenching was not observed upon photobleaching, which may allow for robust photophysics without the need for rapid repair or replacement machinery. Collectively, these observations establish the presence of a rich and robust set of conformational states of PE545. Cryptophytes exhibit particularly diverse energetics owing to the variety of microenvironments in which they survive, and the conformational states and dynamics reported here may provide photophysical flexibility that contributes to their remarkable ability to flourish under diverse conditions.

Identification of fishy odor causing compounds produced by Ochromonas sp. and Cryptomonas ovate with gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography.

Disgusting fishy odor problems have become a major concern in drinking water quality, and are commonly related to algal proliferation in source water. Unlike the typical musty/earthy odorants 2-methylisoborneol (MIB) and geosmin, identification of the corresponding fishy odorants is still a big challenge. In this study, two species of fishy-odor-producing algae, Ochromonas sp. and Cryptomonas ovate, were cultured to explore the odor production characteristics and typical odorants. When algae were ruptured in the stationary and decline phases, fishy odor intensities of 4 to 8 characterized by FPA were produced. However, some frequently reported aldehydes that could cause fishy odor, including n-hexanal, 2-octenal, heptanal, 2,4-heptanal and 2,4-decadienal, were not detected in either of the cultured algae. The possible fishy odor-causing compounds were further identified by combining gas chromatography-olfactometry (GC-O/MS) and comprehensive two-dimensional gas chromatography (GC × GC-TOFMS) using retention indices (RIs). From GC-O/MS analysis, twelve and six olfactometry peaks with various odor characteristics were identified in Ochromonas sp. and Cryptomonas ovate, respectively, of which three and two olfactometry peaks showed fishy odor characteristics. 2-Nonenal, 2,4-octadienal, fluorene and 2-tetradecanone were identified as fishy odorants in Ochromonas sp., and 1-octen-3-ol, 6-methyl-5-hepten-2-one, 1-octen-3-one, 2-nonenal and 2,4-octadienal were identified in Cryptomonas ovate. Other identified compounds, including butyl butanoate (fragrant odor), ionone (fragrant odor), bis (2-chloroisopropyl) ether (chemical odor) etc., did not show fishy features. Therefore, the fishy odor might be a synthetic and comprehensive odor, which resulted from the combination of different odorants and their synergistic effects. The results of this study will be helpful for understanding fishy odor problems, which will provide support for fishy odor management and control in the drinking water industry.

Determination of the protonation preferences of bilin pigments in cryptophyte antenna complexes.

The light-harvesting mechanisms of cryptophyte antenna complexes have attracted considerable attention due to their ability to exhibit maximal photosynthetic activity under very low-light conditions and to display several colors, as well as the observation of vibronic coherent features in their two-dimensional electronic spectra. However, detailed investigations on the interplay between the protein environment and their light-harvesting properties are hampered by the uncertainty related to the protonation state of the underlying bilin pigments. Here we study the protonation preferences of four types of bilin pigments including 15,16-dihydrobiliverdin (DBV), phycoerythrobilin (PEB), phycocyanobilin (PCB) and mesobiliverdin (MBV), which are found in phycoerythrin PE545 and phycocyanin PC577, PC612, PC630 and PC645 complexes. We apply quantum chemical calculations coupled to continuum solvation calculations to predict the intrinsic acidity of bilins in aqueous solution, and then combine molecular dynamics simulations with empirical pKa estimates to investigate the impact of the local protein environment on the acidity of the pigments. We also report measurements of the absorption spectra of the five complexes in a wide range of pH in order to validate our simulations and investigate possible changes in the light harvesting properties of the complexes in the range of physiological pH found in the lumen (pH ∼ 5-7). The results suggest a pKa > 7 for DBV and MBV pigments in the α polypeptide chains of PE545 and PC630/PC645 complexes, which are not coordinated to a negatively charged amino acid. For the other PEB, DBV and PCB pigments, which interact with a Glu or Asp side chain, higher pKa values (pKa > 8) are estimated. Overall, the results support a preferential population of the fully protonated state for bilins in cryptophyte complexes under physiological conditions regardless of the specific type of pigment and local protein environment.

Phycobilins and Phycobiliproteins Used in Food Industry and Medicine.

BACKGROUND: Open tetrapyrroles termed phycobilins represent the major photosynthetic accessory pigments of several cyanobacteria and some eukaryotic algae such as the Glaucophyta, Cryptophyta and Rhodophyta. These pigments are covalently bound to so-called phycobiliproteins which are in general organized into phycobilisomes on the thylakoid membranes. OBJECTIVE & METHODS: In this work we first briefly describe the physico-chemical properties, biosynthesis, occurrence, in vivo localization and roles of the phycobilin pigments and the phycobiliproteins. Then the potential applications and uses of these pigments, pigment-protein complexes and related products by the food industry (e.g., as LinaBlue® or the so-called spirulina extract used as coloring food), by the health industry or as fluorescent dyes are critically reviewed. CONCLUSION: In addition to the stability, bioavailability and safety issues of purified phycobilins and phycobiliproteins, literature data about their antioxidant, anticancer, anti-inflammatory, immunomodulatory, hepatoprotective, nephroprotective and neuroprotective effects, and their potential use in photodynamic therapy (PDT) are also discussed.

Isolation, purification and some ultrastructural details of discharged ejectisomes of cryptophytes.

The first successful isolation of discharged ejectisomes from pigmented cryptophytes is reported. Discharged ejectisomes from a Chroomonas and two Cryptomonas species were characterized by transmission electron microscopy using negative staining and freeze-etching. Tubular-shaped fragments of variable lengths and diameters were obtained which showed a paracrystalline lattice. Particle periodicities of 4.1 nm along the longitudinal axis and 3.1 nm in the transverse direction were measured in negative-stained fragments. The dimensions measured from freeze-etched ejectisome fragments were about 0.5-1 nm larger. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a protein banding pattern, dominated by polypeptides of 40-44, 23-25 and 16-18 kDa. The results are discussed in the context of what is currently known about extrusomes of protists.

Pastas de Rhodomonas salina (Cryptophyta) como alimento para Brachionus plicatilis (Rotifera)

Rhodomonas salina (Cryptophyta) pastes as feed for Brachionus plicatilis (Rotifera). Rotifers are an important live feed for first feeding larvae of many fish species. The use of concentrated algae cells in the mass culture of the rotifer Brachionus plicatilis (Brachionidae) has opened new horizons for research on this organism. Pastes of Rhodomonas salina (Pyrenomonadaceae) obtained either by centrifugation or flocculation with chitosan were preserved, with or without vitamin C, at -20°C for four weeks and were evaluated biochemically (proteins, lipids, pigments and fatty acids contents) and subsequently, were used to feed the rotifer Brachionus plicatilis at a ratio of 25mg/L/day. Four different microalgae pastes were prepared: (1) centrifuged and preserved with vitamin C (CV), (2) centrifuged and preserved without vitamin C (C), (3) flocculated and with vitamin C (FV) and (4) flocculated without vitamin C (F). All treatments showed similar contents of proteins and total lipids with respect to control culture (a fresh culture of R. salina), with mean values of 40.0±2.32% and 12.0±1.45%, respectively. The pheophytin a/chlorophyll a ratio, a general indicator of the chemical status of microalgal concentrates, was similar (0.09-0.11) between centrifuged pastes and control culture, but was found to be higher in flocculated pastes (1.28-1.48). The fatty acid profile varied with respect to the control culture, mainly in the proportion of the essential polyunsaturated fatty acids (PUFAs): eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Total PUFAs, EPA and DHA contents were statistically similar between centrifuged pastes and control culture (PUFAs: 47%, EPA: 4% and DHA: 4.7%), whereas values obtained for flocculated pastes were significantly lower. The rotifers grew equally well when fed with centrifuged pastes or control culture (maximum density: 320rotifers/mL; instantaneous growth rate: 0.23rotifers/day, fecundity: 1.49eggs/female and productivity: 43x103rotifers/L/day. No significant effect of vitamin C was found when used as a paste preservative. We concluded that centrifugation is an effective harvesting method, and that freezing to -20ºC for four weeks (no vitamin added), may help maintain the nutritional quality of R. salina paste, similar to fresh microalgae and can be offered to Brachionus plicatilis. Rev. Biol. Trop. 59 (4): 1503-1515. Epub 2011 December 01.

Pastas de Rhodomonas salina, obtenidas mediante centrifugación y floculación con quitosano y preservadas con o sin vitamina C, a -20°C fueron evaluadas bioquímicamente y proporcionadas como alimento al rotífero Brachionus plicatilis. Las pastas microalgales: (1) centrifugada y con vitamina C (CV), (2) centrifugada y sin vitamina C (C), (3) floculada y con vitamina C (FV) y (4) floculada y sin adición de vitamina C (F); mantuvieron sus contenidos de proteínas y lípidos totales similares al cultivo control, con valores de 40.0±2.32% y 12.0±1.45%, respectivamente. La relación feofitina a/clorofila a fue similar (0.09-0.11) entre las pastas centrifugadas y el cultivo control, pero mayor en las pastas floculadas (1.28-1.48). Las pastas centrifugadas presentaron porcentajes de PUFAs totales, EPA y DHA similares al cultivo control (PUFAs: 47%, EPA: 4% y DHA: 4.7%) y superiores al de las pastas floculadas. Las pastas obtenidas por centrifugación indujeron un crecimiento del rotífero igual al obtenido con el alimento control (densidad máxima: 320rotíferos/mL; tasa instantánea de crecimiento: 0.23rotíferos/día, fecundidad: 1.49huevos/ hembra y productividad: 43x103rotíferos/L/día). Se concluye que la pasta de R. salina centrifugada y congelada a -20°C, durante cuatro semanas, sin adición de vitamina C, mantiene su calidad nutricional similar a la del alga fresca y puede ser usada como alimento de Brachionus plicatilis.

Molecular and biochemical analysis of periplastidial starch metabolism in the cryptophyte Guillardia theta.

Starch in synchronously grown Guillardia theta cells accumulates throughout the light phase, followed by a linear degradation during the night. In contrast to the case for other unicellular algae such as Chlamydomonas reinhardtii, no starch turnover occurred in this organism under continuous light. The gene encoding granule-bound starch synthase (GBSS1), the enzyme responsible for amylose synthesis, displays a diurnal expression cycle. The pattern consisted of a maximal transcript abundance around the middle of the light phase and a very low level during the night. This diurnal regulation of GBSS1 transcript abundance was demonstrated to be independent of the circadian clock but tightly light regulated. A similar yet opposite type of regulation pattern was found for two alpha-amylase isoforms and for one of the two plastidic triose phosphate transporter genes investigated. In these cases, however, the transcript abundance peaked in the night phase. The second plastidic triose phosphate transporter gene had the GBSS1 mRNA abundance pattern. Quantification of the GBSS1 activity revealed that not only gene expression but also total enzyme activity exhibited a maximum in the middle of the light phase. To gain a first insight into the transport processes involved in starch biosynthesis in cryptophytes, we demonstrated the presence of both plastidic triose phosphate transporter and plastidic ATP/ADP transporter activities in proteoliposomes harboring either total membranes or plastid envelope membranes from G. theta. These molecular and biochemical data are discussed with respect to the environmental conditions experienced by G. theta and with respect to the unique subcellular location of starch in cryptophytes.