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.

Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel.

The crystal structure of the light-gated anion channel GtACR1 reported in our previous Research Article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool.

Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2" from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.

Quantum dynamics of vibration-assisted excitation energy transfer in phycobiliprotein light-harvesting complex.

Phycobiliprotein is a light-harvesting complex containing linear tetrapyrrole bilin pigments that are responsible for absorption and funneling the sun's energy in cryptophytes algae. In particular, the protein structure determines relative positions and orientations of the pigments and thus controls energy transfer pathways. The present research reveals the impact of molecular vibrations (in the 850-2700 cm-1 region) on excitation energy transfer in phycobiliprotein. The analysis of the excitation energy transfer pathways indicates a possibility of the coherent mechanism of energy transfer (delocalization) in central dihydrobiliverdin pigments and incoherent vibration-assisted energy transfer to peripheral phycocyanobilin pigments at a sub-picosecond time scale. A computational approach that enables modeling the dynamics of the excitation energy transfer with the quantum master equation formalism employing Huang-Rhys factors to describe electronic-vibrational coupling has been developed. The computational methodology has been implemented in PyFREC software.

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.

Influence of the Protein Environment on the Electronic Excitation of Chromophores in the Phycoerythrin 545 Light-Harvesting Complex: A Combined MD-QM/MM Method with Polarized Protein-Specific Charge Scheme.

To gain better insight into how the fluctuating protein environment influences the site energy ordering of the chromophores in PE545 light-harvesting antenna system, we carried out quantum mechanics/molecular mechanics (QM/MM) calculations along the molecular dynamics (MD) trajectory. The Polarized Protein-Specific Charge (PPC) scheme was adopted in both the MD simulation and the QM/MM calculations for a more realistic description of the protein environment. The deduced site energy ladder calculated using ZINDO/S-CIS agrees well with the best model extracted from experiments by a simultaneous fit of the steady-state spectra and transient absorption spectra. Three combinations of charge schemes were compared to elucidate how the protein environment modulates the site energy of chromophores. The result indicates that the multiroles that the protein environment is playing, for instance, by fine-tuning of the conformation of chromophores or by specific pigment-protein interactions, are both crucial for site energy arrangement. Furthermore, we investigated the effects of individual environments and found that the polar residues and water molecules contribute most to the energy shifts.

Prey Lysate Enhances Growth and Toxin Production in an Isolate of Dinophysis acuminata.

The physiological and toxicological characteristics of Dinophysis acuminata have been increasingly studied in an attempt to better understand and predict diarrhetic shellfish poisoning (DSP) events worldwide. Recent work has identified prey quantity, organic nitrogen, and ammonium as likely contributors to increased Dinophysis growth rates and/or toxicity. Further research is now needed to better understand the interplay between these factors, for example, how inorganic and organic compounds interact with prey and a variety of Dinophysis species and/or strains. In this study, the exudate of ciliate prey and cryptophytes were investigated for an ability to support D. acuminata growth and toxin production in the presence and absence of prey, i.e., during mixotrophic and phototrophic growth respectively. A series of culturing experiments demonstrated that the addition of ciliate lysate led to faster dinoflagellate growth rates (0.25 ± 0.002/d) in predator-prey co-incubations than in treatments containing (1) similar levels of prey but without lysate (0.21 ± 0.003/d), (2) ciliate lysate but no live prey (0.12 ± 0.004/d), or (3) monocultures of D. acuminata without ciliate lysate or live prey (0.01 ± 0.007/d). The addition of ciliate lysate to co-incubations also resulted in maximum toxin quotas and extracellular concentrations of okadaic acid (OA, 0.11 ± 0.01 pg/cell; 1.37 ± 0.10 ng/mL) and dinophysistoxin-1 (DTX1, 0.20 ± 0.02 pg/cell; 1.27 ± 0.10 ng/mL), and significantly greater total DSP toxin concentrations (intracellular + extracellular). Pectenotoxin-2 values, intracellular or extracellular, did not show a clear trend across the treatments. The addition of cryptophyte lysate or whole cells, however, did not support dinoflagellate cell division. Together these data demonstrate that while certain growth was observed when only lysate was added, the benefits to Dinophysis were maximized when ciliate lysate was added with the ciliate inoculum (i.e., during mixotrophic growth). Extrapolating to the field, these culturing studies suggest that the presence of ciliate exudate during co-occurring dinoflagellate-ciliate blooms may indirectly and directly exacerbate D. acuminata abundance and toxigenicity. More research is required, however, to understand what direct or indirect mechanisms control the predator-prey dynamic and what component(s) of ciliate lysate are being utilized by the dinoflagellate or other organisms (e.g., ciliate or bacteria) in the culture if predictive capabilities are to be developed and management strategies created.

Revealing structural involvement of chromophores in algal light harvesting complexes using symmetry-adapted perturbation theory.

The attribution of quantum beats observed in the time-resolved spectroscopy of photosynthetic light-harvesting antennae to nontrivial quantum coherences has sparked a flurry of research activity beginning a decade ago. Even though investigations into the functional aspects of photosynthetic light-harvesting were supported by X-ray crystal structures, the non-covalent interactions between pigments and their local protein environment that drive such function has yet to be comprehensively explored. Using symmetry-adapted perturbation theory (SAPT), we have comprehensively determined the magnitude and compositions of these non-covalent interactions involving light-harvesting chromophores in two quintessential photosynthetic pigment-protein complexes - peridinin chlorophyll-a protein (PCP) from dinoflagellate Amphidinium carterae and phycocyanin 645 (PC645) from cryptophyte Chroomonas mesostigmatica. In PCP, the chlorophylls are dispersion-bound to the peridinins, which in turn are electrostatically anchored to the protein scaffold via their polar terminal rings. This might be an evolutionary design principle in which the relative orientation of the carotenoids towards the aqueous environment determines the arrangement of the other chromophores in carotenoid-based antennas. On the other hand, electrostatics dominate the non-covalent interactions in PC645. Our ab initio simulations also suggest full protonation of the PC645 chromophores in physiological conditions, and that changes to their protonation states result in their participation as switches between folded and unfolded conformations.

Epiplasts: Membrane Skeletons and Epiplastin Proteins in Euglenids, Glaucophytes, Cryptophytes, Ciliates, Dinoflagellates, and Apicomplexans.

Animals and amoebae assemble actin/spectrin-based plasma membrane skeletons, forming what is often called the cell cortex, whereas euglenids and alveolates (ciliates, dinoflagellates, and apicomplexans) have been shown to assemble a thin, viscoelastic, actin/spectrin-free membrane skeleton, here called the epiplast. Epiplasts include a class of proteins, here called the epiplastins, with a head/medial/tail domain organization, whose medial domains have been characterized in previous studies by their low-complexity amino acid composition. We have identified two additional features of the medial domains: a strong enrichment of acid/base amino acid dyads and a predicted ß-strand/random coil secondary structure. These features have served to identify members in two additional unicellular eukaryotic radiations-the glaucophytes and cryptophytes-as well as additional members in the alveolates and euglenids. We have analyzed the amino acid composition and domain structure of 219 epiplastin sequences and have used quick-freeze deep-etch electron microscopy to visualize the epiplasts of glaucophytes and cryptophytes. We define epiplastins as proteins encoded in organisms that assemble epiplasts, but epiplastin-like proteins, of unknown function, are also encoded in Insecta, Basidiomycetes, and Caulobacter genomes. We discuss the diverse cellular traits that are supported by epiplasts and propose evolutionary scenarios that are consonant with their distribution in extant eukaryotes.IMPORTANCE Membrane skeletons associate with the inner surface of the plasma membrane to provide support for the fragile lipid bilayer and an elastic framework for the cell itself. Several radiations, including animals, organize such skeletons using actin/spectrin proteins, but four major radiations of eukaryotic unicellular organisms, including disease-causing parasites such as Plasmodium, have been known to construct an alternative and essential skeleton (the epiplast) using a class of proteins that we term epiplastins. We have identified epiplastins in two additional radiations and present images of their epiplasts using electron microscopy. We analyze the sequences and secondary structure of 219 epiplastins and present an in-depth overview and analysis of their known and posited roles in cellular organization and parasite infection. An understanding of epiplast assembly may suggest therapeutic approaches to combat infectious agents such as Plasmodium as well as approaches to the engineering of useful viscoelastic biofilms.

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.

Crystal structure of the natural anion-conducting channelrhodopsin GtACR1.

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Baffinella frigidus gen. et sp. nov. (Baffinellaceae fam. nov., Cryptophyceae) from Baffin Bay: Morphology, pigment profile, phylogeny, and growth rate response to three abiotic factors.

Twenty years ago an Arctic cryptophyte was isolated from Baffin Bay and given strain number CCMP2045. Here, it was described using morphology, water- and non-water soluble pigments and nuclear-encoded SSU rDNA. The influence of temperature, salinity, and light intensity on growth rates was also examined. Microscopy revealed typical cryptophyte features but the chloroplast color was either green or red depending on the light intensity provided. Phycoerythrin (Cr-PE 566) was only produced when cells were grown under low-light conditions (5 µmol photons · m-2  · s-1 ). Non-water-soluble pigments included chlorophyll a, c2 and five major carotenoids. Cells measured 8.2 × 5.1 µm and a tail-like appendage gave them a comma-shape. The nucleus was located posteriorly and a horseshoe-shaped chloroplast contained a single pyrenoid. Ejectosomes of two sizes and a nucleomorph anterior to the pyrenoid were discerned in TEM. SEM revealed a slightly elevated vestibular plate in the vestibulum. The inner periplast component consisted of slightly overlapping hexagonal plates arranged in 16-20 oblique rows. Antapical plates were smaller and their shape less profound. Temperature and salinity studies revealed CCMP2045 as stenothermal and euryhaline and growth was saturated between 5 and 20 µmol photons · m-2  · s-1 . The phylogeny based on SSU rDNA showed that CCMP2045 formed a distinct clade with CCMP2293 and Falcomonas sp. isolated from Spain. Combining pheno- and genotypic data, the Arctic cryptophyte could not be placed in an existing family and genus and therefore Baffinellaceae fam. nov. and Baffinella frigidus gen. et sp. nov. were proposed.

On-line analysis of algae in water by discrete three-dimensional fluorescence spectroscopy.

In view of the problem of the on-line measurement of algae classification, a method of algae classification and concentration determination based on the discrete three-dimensional fluorescence spectra was studied in this work. The discrete three-dimensional fluorescence spectra of twelve common species of algae belonging to five categories were analyzed, the discrete three-dimensional standard spectra of five categories were built, and the recognition, classification and concentration prediction of algae categories were realized by the discrete three-dimensional fluorescence spectra coupled with non-negative weighted least squares linear regression analysis. The results show that similarities between discrete three-dimensional standard spectra of different categories were reduced and the accuracies of recognition, classification and concentration prediction of the algae categories were significantly improved. By comparing with that of the chlorophyll a fluorescence excitation spectra method, the recognition accuracy rate in pure samples by discrete three-dimensional fluorescence spectra is improved 1.38%, and the recovery rate and classification accuracy in pure diatom samples 34.1% and 46.8%, respectively; the recognition accuracy rate of mixed samples by discrete-three dimensional fluorescence spectra is enhanced by 26.1%, the recovery rate of mixed samples with Chlorophyta 37.8%, and the classification accuracy of mixed samples with diatoms 54.6%.

Marine Cryptophytes Are Great Sources of EPA and DHA.

Microalgae have the ability to synthetize many compounds, some of which have been recognized as a source of functional ingredients for nutraceuticals with positive health effects. One well-known example is the long-chain polyunsaturated fatty acids (PUFAs), which are essential for human nutrition. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the two most important long-chain omega-3 (ω-3) PUFAs involved in human physiology, and both industries are almost exclusively based on microalgae. In addition, algae produce phytosterols that reduce serum cholesterol. Here we determined the growth rates, biomass yields, PUFA and sterol content, and daily gain of eight strains of marine cryptophytes. The maximal growth rates of the cryptophytes varied between 0.34-0.70 divisions day-1, which is relatively good in relation to previously screened algal taxa. The studied cryptophytes were extremely rich in ω-3 PUFAs, especially in EPA and DHA (range 5.8-12.5 and 0.8-6.1 µg mg dry weight-1, respectively), but their sterol concentrations were low. Among the studied strains, Storeatula major was superior in PUFA production, and it also produces all PUFAs, i.e., α-linolenic acid (ALA), stearidonic acid (SDA), EPA, and DHA, which is rare in phytoplankton in general. We conclude that marine cryptophytes are a good alternative for the ecologically sustainable and profitable production of health-promoting lipids.

The effect of algae diets (Skeletonema costatum and Rhodomonas baltica) on the biochemical composition and sensory characteristics of Pacific cupped oysters (Crassostrea gigas) during land-based refinement.

Oyster refinement, a common practice in France, is aimed at increasing the weight of oyster tissue and influencing the taste properties of the refined oysters. Refinement usually takes place in land-based systems where the oysters are fed with relatively high concentrations of microalgae. In this study the impact of feeding Skeletonema costatum and Rhodomonas baltica on the biochemical composition and sensory characteristics of Pacific cupped oysters (Crassostrea gigas) from the Eastern Scheldt during land-based refinement was studied. After a feeding period of four and seven weeks market-sized oysters were sampled for the analysis of fatty acids, free amino acids and volatile organic compounds and for a sensory evaluation by consumers and an expert panel. The algae Skeletonema costatum showed a lower ∑PUFA, ∑n-3, ∑n-6, C18:2n6, C18:3n3, C18:4n3, C22:6n3 content as compared with Rhodomonas baltica. These differences were also reflected in the fatty acid profile of the oysters fed with the corresponding algae diets. Furthermore, general linear model and principal component analysis showed marked differences in free amino acids and volatile organic compound content between Skeletonema, Rhodomonas fed oysters and reference oysters. For example, threonine, glutamine, leucine, histidine, (E)-2-hexenal, (E)-2octenal, (E)-2-octen-1-ol, (E,E)-2,4-octadien-1-ol, (E,Z)-3,6-nonadien-1-ol and (Z,E)-2,6-nonadienal contents were higher in Skeletonema fed oysters compared to Rhodomonas fed oysters. Sensory differences between the experimental oyster groups were shown. Skeletonema fed Pacific cupped oysters were characterized by a stronger seaweed flavor, higher perceived sweetness and a firmer texture in comparison with Rhodomonas fed oysters. Naïve consumers were only able to differentiate between Rhodomonas fed oysters and reference oysters.

First-Principles Models for Biological Light-Harvesting: Phycobiliprotein Complexes from Cryptophyte Algae.

There have been numerous efforts, both experimental and theoretical, that have attempted to parametrize model Hamiltonians to describe excited state energy transfer in photosynthetic light harvesting systems. The Frenkel exciton model, with its set of electronically coupled two level chromophores that are each linearly coupled to dissipative baths of harmonic oscillators, has become the workhorse of this field. The challenges to parametrizing such Hamiltonians have been their uniqueness, and physical interpretation. Here we present a computational approach that uses accurate first-principles electronic structure methods to compute unique model parameters for a collection of local minima that are sampled with molecular dynamics and QM geometry optimization enabling the construction of an ensemble of local models that captures fluctuations as these systems move between local basins of inherent structure. The accuracy, robustness, and reliability of the approach is demonstrated in an application to the phycobiliprotein light harvesting complexes from cryptophyte algae. Our computed Hamiltonian ensemble provides a first-principles description of inhomogeneous broadening processes, and a standard approximate non-Markovian reduced density matrix dynamics description is used to estimate lifetime broadening contributions to the spectral line shape arising from electronic-vibrational coupling. Despite the overbroadening arising from this approximate line shape theory, we demonstrate that our model Hamiltonian ensemble approach is able to provide a reliable fully first-principles method for computation of spectra and can distinguish the influence of different chromophore protonation states in experimental results. A key feature in the dynamics of these systems is the excitation of intrachromophore vibrations upon electronic excitation and energy transfer. We demonstrate that the Hamiltonian ensemble approach provides a reliable first-principles description of these contributions that have been detailed in recent broad-band pump-probe and two-dimensional electronic spectroscopy experiments.

The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.

Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl- and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl--conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated "ZipACR", is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2-4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency.

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.

In Vitro Activity of a Purified Natural Anion Channelrhodopsin.

Natural anion channelrhodopsins (ACRs) recently discovered in cryptophyte algae are the most active rhodopsin channels known. They are of interest both because of their unique natural function of light-gated chloride conductance and because of their unprecedented efficiency of membrane hyperpolarization for optogenetic neuron silencing. Light-induced currents of ACRs have been studied in HEK cells and neurons, but light-gated channel conductance of ACRs in vitro has not been demonstrated. Here we report light-induced chloride channel activity of a purified ACR protein reconstituted in large unilamellar vesicles (LUVs). EPR measurements establish that the channels are inserted uniformly "inside-out" with their cytoplasmic surface facing the medium of the LUV suspension. We show by time-resolved flash spectroscopy that the photochemical reaction cycle of a functional purified ACR from Guillardia theta (GtACR1) in LUVs exhibits similar spectral shifts, indicating similar photocycle intermediates as GtACR1 in detergent micelles. Furthermore, the photocycle rate is dependent on electric potential generated by chloride gradients in the LUVs in the same manner as in voltage-clamped animal cells. We confirm with this system that, in contrast to cation-conducting channelrhodopsins, opening of the channel occurs prior to deprotonation of the Schiff base. However, the photointermediate transitions in the LUVs exhibit faster kinetics. The ACR-incorporated LUVs provide a purified defined system amenable to EPR, optical and vibrational spectroscopy, and fluorescence resonance energy transfer measurements of structural changes of ACRs with the molecules in a demonstrably functional state.