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Introduction: Cyanobacterium Arthrospira platensis (AP) (Nordstedt) Gomont contains high content of phycobiliproteins (PBP), which are an important source for food industry. Methods effectively extracting proteins contained in AP cells are demanded to provide a supply of the material. Water-based extraction methods are advisable due to the high solubility of the PBP., Objectives: Extraction techniques such as ultrasound assisted extraction (UAE) and pressurized water extraction (PWE) are popular due to their environmental friendliness, better extraction efficiency, and faster extraction process. In this paper, efficiency of the two methods is compared., Materials and Methods: PWE along with UAE is utilized for release of PBP from the AP cells. The extraction parameters including time, temperature, pressure, and ultrasound intensity are tested to obtain the most efficient setup. The methods were evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the replicates of PWE extracts were further analyzed by capillary isoelectric focusing with laser-induced fluorescence (cIEF-LIF)., Results: The developed PWE method using higher pressure treatment at lower temperature was significantly faster than UAE methods, and the SDS-PAGE results showed a high content of phycobiliproteins in the extracts. cIEF-LIF analysis showed that the sequential PWE of individual samples was repeatable, and the mild extraction provided a fluorescent profile similar to the commercially available C-phycocyanin standard., Conclusion: Pressurized water extraction was shown to be an efficient, rapid, and well-automated extraction method for AP proteins in general, including bioactive phycobiliproteins. Obtained results encourage the use of PWE in small-scale analytical applications for primary extraction of proteins., (© 2024 The Author(s). Phytochemical Analysis published by John Wiley & Sons Ltd.)

The food industry is increasingly turning to microalgae pigments as natural functional colourants to replace potentially harmful synthetic dyes. Among those, phycobiliproteins offer deep and vibrant colours with bioactivities, showing to be a promising alternative. However, incorporating them into a food matrix can be challenging due to their inherent instability, low bioavailability, and off-flavours. Accordingly, the present work aimed to prepare microparticles of phycocyanin (PC MP ) and of an ultrasound-assisted phycoerythrin-rich extract from Porphyridium purpureum (PE MP ) to improve these natural colourants' stability and functional properties. The microparticles were prepared by Nano spray-drying using inulin and gum Arabic as coating materials, resulting in particles with high yield, encapsulation efficiency (from 69 % to 80 %), solubility, and colour retention. Combining inulin and gum Arabic allowed the formation of smaller and more homogeneous particles with increased antioxidant potential when compared to using inulin alone. Moreover, gum Arabic improved the bioaccessibility of PE MP from 35 % to 51 %, demonstrating the potential of this microparticle formulation to enhance the stability and functionality of phycobiliproteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Arthrospira platensis has been utilized as a food source since ancient times due to its rich nutrient profile. In recent years, its popularity as a dietary supplement has soared, especially due to the presence of a water-soluble phycobiliprotein, C-phycocyanin C (C-PC), which is abundant and notable for its fluorescent properties. C-PC contains the chromophore phycocyanobilin B (PCB-B), a tetrapyrrole molecule, that is why it plays a dual role as a food colorant and as nutraceutical. However, comprehensive studies have mostly evaluated C-PC's broader health-promoting properties, particularly its antioxidative and anti-inflammatory effects, which are linked to its ability to contrast oxidative stress and related pathological conditions. That is why this review explores recent advancements in optimizing culture conditions to enhance C-PC and PCB-B production, with a particular emphasis on novel extraction and purification techniques that increase yield and bioactivity. This focus on efficient production methods is crucial for expanding the commercial and therapeutic applications of C-PC, contributing to its growing relevance in the food and pharmaceutical industries., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A biorefinery process was developed to isolate phycobiliproteins, sulfated polysaccharides, and phenolic compounds from Palmaria palmata . The extraction process was carried out in three stages using ultrasound-assisted extraction (UAE) and pressurized liquid extraction (PLE) integrated with different natural deep eutectic solvents (NaDESs). In general, PLE provided higher phycobiliprotein contents than UAE in the first step of the process. In fact, the hydrolysis product of the PLE-NaDES extracts achieved a higher antioxidant capacity than that of the UAE-NaDES extracts. Particularly, glycerol:glucose (2:1) with 50% water in combination with PLE was the most suitable NaDES to recover the highest phycobiliprotein, protein, and sulfated polysaccharide contents from Palmaria palmata in the first and second steps of the biorefinery process. Finally, a PLE-NaDES using choline chloride:glycerol (1:2) with 60% water as the NaDES was employed for the recovery of antioxidant and neuroprotective phenolic compounds from the residue of the second step, obtaining a higher total phenolic content than employing PLE with ethanol/water (70:30, v / v ) as the extraction solvent. Moreover, a forced stability study revealed that the NaDESs provided a protective effect compared to the water extracts against the degradation of phycobiliproteins, preserving their color over time. This study contributes to the recovery of high-value components from an undervalued biomarine source through a sustainable biorefinery process., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in th1.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products., Competing Interests: Declaration of competing interest Jeongmi Lee reports financial support was provided by National Research Foundation of Korea. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Arthrospira maxima is a source of phycobiliproteins with different nutraceutical properties, e.g. antioxidant and anti-inflammatory activities. The current study was aimed at the elaboration, characterization, and evaluation of the anti-inflammatory effect of the phycobiliprotein nanoparticles extracted from Arthrospira maxima. Previously freeze-dried phycobiliproteins were milled by high-energy ball milling until reaching a nanometric size (optimal time: 4 h). Microscopy techniques were used for the characterization of the size and morphology of phycobiliproteins nanoparticles. Additionally, a spectroscopic study evidenced that nanosized reduction induced an increase in the chemical functional groups associated with its anti-inflammatory activity that was tested in a murine model, showing an immediate inflammatory effect. The novelty and importance of this contribution was to demonstrate that high energy ball milling is an emerging and green technology that can produce phycobiliprotein nanoparticles on a large-scale, without the use of organic solvents, to test their nutraceutical properties in a biological model by intragastric administration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microalgae are a source of a wide variety of commodities, including particularly valuable pigments. The typical pigments present in microalgae are the chlorophylls, carotenoids, and phycobiliproteins. However, other types of pigments, of the family of water-soluble polyphenols, usually encountered in terrestrial plants, have been recently reported in microalgae. Among such microalgal polyphenols, many flavonoids have a yellowish hue, and are used as natural textile dyes. Besides being used as natural colorants, for example in the food or cosmetic industry, microalgal pigments also possess many bioactive properties, making them functional as nutraceutical or pharmaceutical agents. Each type of pigment, with its own chemical structure, fulfills particular biological functions. Considering both eukaryotes and prokaryotes, some species within the four most promising microalgae groups (Cyanobacteria, Rhodophyta, Chlorophyta and Heterokontophyta) are distinguished by their high contents of specific added-value pigments. To further enhance microalgae pigment contents during autotrophic cultivation, a review is made of the main related strategies adopted during the last decade, including light adjustments (quantity and quality, and the duration of the photoperiod cycle), and regard to mineral medium characteristics (salinity, nutrients concentrations, presence of inductive chemicals). In contrast to what is usually observed for growth-related pigments, accumulation of non-photosynthetic pigments (polyphenols and secondary carotenoids) requires particularly stressful conditions. Finally, pigment enrichment is also made possible with two new cutting-edge technologies, via the application of metallic nanoparticles or magnetic fields., (© 2024. The Author(s).)

Vivid-colored phycobiliproteins (PBPs) have emerging potential as food colors and alternative proteins in the food industry. However, enhancing their application potential requires increasing stability, cost-effective purification processes, and consumer acceptance. This narrative review aimed to highlight information regarding the critical aspects of PBP research that is needed to improve their food industry potential, such as stability, food fortification, development of new PBP-based food products, and cost-effective production. The main results of the literature review show that polysaccharide and protein-based encapsulations significantly improve PBPs' stability. Additionally, while many studies have investigated the ability of PBPs to enhance the techno-functional properties, like viscosity, emulsifying and stabilizing activity, texture, rheology, etc., of widely used food products, highly concentrated PBP food products are still rare. Therefore, much effort should be invested in improving the stability, yield, and sensory characteristics of the PBP-fortified food due to the resulting unpleasant sensory characteristics. Considering that most studies focus on the C-phycocyanin from Spirulina, future studies should concentrate on less explored PBPs from red macroalgae due to their much higher production potential, a critical factor for positioning PBPs as alternative proteins.

Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190 % in vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180 % in vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy., (© 2024 Wiley-VCH GmbH.)

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700-800 nm) than the wavelengths typically used for oxygenic photosynthesis (400-700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-EM to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently, the use of synthetic pigments in foods is restricted since synthetic pigments are proven and suspected to be harmful to human health. Phycobiliproteins (PBPs), existed in phycobilisomes (PBSs) of algae, are a kind of pigment-proteins with intense color. The specific color of PBPs (red and blue) is given by the water-soluble open-chained tetrapyrrole chromophore (phycobilin) that covalently attaches to the apo-protein via thioether linkages to cysteine residues. According to the spectral characteristics of PBPs, they can be categorized as phycoerythrins (PEs), phycocyanins (PCs), allophycocyanins (APCs), and phycoerythrocyanins (PECs). PBPs can be used as natural food colorants, fluorescent substances, and bioactive ingredients in food applications owing to their color characteristics and physiological activities. This paper mainly summarizes the extraction and purification methods of the PBPs and reviews their characteristics and applications. Moreover, the use of several strategies such as additives, microencapsulation, electrospray, and cross-linking to improve the stability and bioavailability of PBPs as well as the future outlooks of PBPs as natural colorants in food commercialization are elucidated.

Algae are exclusively aquatic photosynthetic organisms that are microscopic or macroscopic, unicellular or multicellular and distributed across the globe. They are a potential source of food, feed, medicine and natural pigments. A variety of natural pigments are available from algae including chlorophyll a, b, c d, phycobiliproteins, carotenes and xanthophylls. The xanthophylls include acyloxyfucoxanthin, alloxanthin, astaxanthin, crocoxanthin, diadinoxanthin, diatoxanthin, fucoxanthin, loroxanthin, monadoxanthin, neoxanthin, nostoxanthin, perdinin, Prasinoxanthin, siphonaxanthin, vaucheriaxanthin, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, while carotenes include echinenone, α-carotene, β-carotene, γ-carotene, lycopene, phytoene, phytofluene. These pigments have applications as pharmaceuticals and nutraceuticals and in the food industry for beverages and animal feed production. The conventional methods for the extraction of pigments are solid-liquid extraction, liquid-liquid extraction and soxhlet extraction. All these methods are less efficient, time-consuming and have higher solvent consumption. For a standardized extraction of natural pigments from algal biomass advanced procedures are in practice which includes Supercritical fluid extraction, Pressurized liquid extraction, Microwave-assisted extraction, Pulsed electric field, Moderate electric field, Ultrahigh pressure extraction, Ultrasound-assisted extraction, Subcritical dimethyl ether extraction, Enzyme assisted extraction and Natural deep eutectic solvents. In the present review, these methods for pigment extraction from algae are discussed in detail.

Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αβ protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one β subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein., (© 2023. The Author(s).)

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

Aquatic photosynthetic organisms evolved to use a variety of light frequencies to perform photosynthesis. Phycobiliprotein phycocyanin 645 (PC645) is a light-harvesting complex in cryptophyte algae able to transfer the absorbed green solar light to other antennas with over 99% efficiency. The infrared signatures of the phycobilin pigments embedded in PC645 are difficult to access and could provide useful information to understand the mechanism behind the high efficiency of energy transfer in PC645. We use visible-pump IR-probe and two-dimensional electronic vibrational spectroscopy to study the dynamical evolution and assign the fingerprint mid-infrared signatures to each pigment in PC645. Here, we report the pigment-specific vibrational markers that enable us to track the spatial flow of excitation energy between the phycobilin pigment pairs. We speculate that two high-frequency modes (1588 and 1596 cm -1 ) are involved in the vibronic coupling leading to fast (

Blue-green algae, also known as cyanobacteria, contain some of the most efficient light-harvesting complexes known. These large, colourful complexes consist of phycobiliproteins which are extremely valuable in the cosmetics, food, nutraceutical and pharmaceutical industries. Additionally, the colourful and fluorescent properties of phycobiliproteins can be modulated by metal ions, making them highly attractive as heavy metal sensors and heavy metal scavengers. Although the overall quenching ability metal ions have on phycobiliproteins is known, the mechanism of heavy metal binding to phycobiliproteins is not fully understood, limiting their widespread quantitative applications. Here, we show using high-resolution native mass spectrometry that phycobiliprotein complexes bind metal ions in different manners. Through monitoring the binding equilibria and metal-binding stoichiometry, we show in particular copper and silver to have drastic, yet different effects on phycobiliprotein structure, both copper and silver modulate the overall complex properties. Together, the data reveals the mechanisms by which metal ions can modulate phycobiliprotein properties which can be used as a basis for the future design of metal-related phycobiliprotein applications., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Attachment of bilins to phycobiliproteins is performed by dedicated lyases. In this issue of Structure, Kumarapperuma et al., 2022 present the structure of an E/F type lyase-isomerase that identifies the correct biological interface between active domains, suggesting that a previous E/F lyase misidentified the heterodimer structure from the crystal lattice., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Phycobiliprotein is a natural product with many biological activities in various seaweeds. Phycobiliproteins have been widely used for anti-oxidation, anti-tumor, anti-inflammatory and immune-enhancing activities as a functional factor. Phycobiliproteins with high purity are considerably more expensive than common. To provide with a systematic, deep and detailed information about those features of phycobiliproteins, we performed a relatively comprehensive analysis on structural composition, the application of phycobiliproteins in the fields of fluorescent probe and photodynamic therapy in this report., (Copyright © 2021. Published by Elsevier B.V.)

Cryptophytes are among the few eukaryotes employing phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to cyanobacterial PBP that are organized in membrane-associated phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin 545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. The assembly of PBPs in cryptophytes involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of the eukaryotic S-type PBP lyase GtCPES. We show GtCPES-mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP β-subunit (PmCpeB) of Prochlorococcus marinus MED4. On the basis of the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB), most likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colored complex in vitro and produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on these findings, we postulate that this single amino acid residue has a crucial role for bilin binding specificity of S-type phycoerythrin lyases but additional factors regulate handover to the target protein., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study ascertained the stability of phycobiliprotein (PBP), a bioactive protein from Dulse (Palmaria palmata) loaded within liposomes and stabilized with polyethylene glycol (2000 and 4000 g/mol) and desulfated CNCs (DCs) containing adsorbed polyethylene glycol (DCs-2000 and DCs-4000). The effect of pH, temperature and illumination on the stability of PBP was investigated. Results showed that the temperature had the most significant (p < 0.05) effect on the fluorescence intensity of the PBP, accounting for up to 70% loss of the fluorescence intensity for PBP loaded liposome (PL), PL stabilized with PEG-2000 (PLP-2000) and PEG 4000 (PLP-4000) and PL stabilized with desulfated CNCs (DCs), however, 60% for the PL stabilized with PEG 2000 and PEG 4000 adsorbed CNCs (PLDCs-2000 and PLDCs-4000) at 60 °C compared to those stabilized at 4 °C. A further increase in temperature to 80 °C led to a complete loss of fluorescence. Operating at the extreme pH's of 1.0 and 11.0 resulted in a loss of 90% and 30% fluorescence intensity, respectively for PBP in solution, whereas, 20% and 2% loss was observed for PBP incorporated inside the liposomes. Regarding the effect of illumination, PLDCs-2000 and PLDCs-4000 were the most stable, retaining the fluorescence intensity of PBP up to 70% after 72 h of exposure. This is compared to 85% loss of fluorescence for PBP in solution. Furthermore, at pH of 1.0, there was an increase in average particle size for the PLDCs-2000 and PLDCs-4000 from 189 ± 3 & 206 ± 2 nm to 6464 ± 211 & 6698 ± 317 nm and a charge reversal in the zeta potential from -36 ± 1 & -34 ± 2 to +16 ± 3 & +14 ± 1. Confocal and optical microscopic images confirmed the coalescence of PBP loaded liposome and agglomeration PLDCs-2000 and PLDCs-4000 under acidic pH (<3.0). In contrast, changes in temperature from 4 °C to 100 °C and illumination as a function of time up to 72 h resulted in no change in liposome size and zeta potential., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phycobiliprotein (PBP) pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer using previously optimized conditions. The stability of PBPs in the extracts was assessed by monitoring the extracts at different pHs and temperatures for 10 days. Since phycoerythrin (PE) is the main PBP present in G. gracilis, PE content was spectroscopically determined and used as a response factor. Kinetic modeling was used to describe PE degradation under different ranges of T and pH. The pigment extracts presented higher stability at pH 6.9 and -20 °C. PE was semipurified by precipitation with ammonium sulphate 65% followed by dialysis against water until a purity index of 0.7. The pigment was successfully applied as colorant in pancakes and yogurts with a pigment concentration of 0.15%. This study highlights the potential of PE pigments extracted from G. gracilis for applications in food products. PRACTICAL APPLICATION: Phycobiliprotein pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer. The stability of the pigment was evaluated at different pHs and temperatures, presenting higher stability at neutral pH and low temperatures. The pigment was successfully applied as colorant in pancakes and yogurts with a low pigment concentration. This study highlights the potential of phycobiliprotein pigments extracted from G. gracilis for applications in food products., (© 2020 Institute of Food Technologists®.)

The freezing-thawing method had been reported to be the best phycobiliprotein extraction technique. However, optimum parameters of this extraction method for Arthrospira sp. (one of the major phycobiliprotein sources) still remained unclear. Hence, this study aimed to optimize the freezing-thawing parameters of phycobiliprotein extraction in Arthrospira sp. (UPMC-A0087). The optimization of the freezing-thawing method was conducted using different solvents, biomass/solvent ratios, temperatures, time intervals and freezing-thawing cycles. The extracted phycobiliproteins were quantified using a spectrophotometric assay. Double distilled water (pH 7) with a 0.50% w / v biomass/solvent ratio was the most efficient solvent in extracting high concentrations and purity of phycobiliproteins from Arthrospira sp. In addition, the combination of freezing at -80 °C (2 h) and thawing at 25 °C (24 h) appeared to be the optimum temperature and extraction time to obtain the highest amount of phycobiliproteins. A minimum of one cycle of freezing and thawing was sufficient for extracting high concentrations of phycobiliproteins. The findings from this study could reduce the cost and labor needed for extracting high quality phycobiliproteins. It also allowed the harvesting of large amounts of valuable phycobiliproteins.

The extraction of phycobiliprotein (PBP) pigments from red algae Gracilaria gracilis was optimized using maceration, ultrasound-assisted extraction (ultrasonic water bath and ultrasonic probe), high pressure-assisted extraction, and freeze-thaw. The experimental conditions, namely homogenization time (t1), buffer concentration (C), treatment time (t2), biomass: buffer ratio (R), and pressure (P), were optimized using Response Surface Methodology (RSM). The yield of phycoerythrin (PE) extracted, determined spectroscopically, was used as the response variable. Maceration was the most efficient extraction method yielding 3.6 mg PE/g biomass under the optimal conditions (t1 = t2 = 10 min; C = 0.1 M; R = 1:50). Scanning Electron Microscopy (SEM) analysis of the biomass before and after the cell disruption treatments revealed a more efficient cell wall rupture with maceration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Arthrospira platensis (spirulina) is a cyanobacterium, which contains mainly two phycobiliproteins (PBP), i.e., C-phycocyanin (C-PC) and allophycocyanin (APC). In this study, PBP were hydrolyzed using trypsin, and the composition of the hydrolysate was characterized by HPLC-ESI-MS/MS. Furthermore, the potential anti-diabetic activity was assessed by using either biochemical or cellular techniques. Findings suggest that PBP peptides inhibit DPP-IV activity in vitro with a dose-response trend and an IC 50 value falling in the range between 0.5 and 1.0 mg/mL. A lower inhibition of the DPP-IV activity expressed by Caco-2 cells was observed, which was explained by a secondary metabolic degradation exerted by the same cells.

Cyanobacteria are photosynthetic microorganisms that are considered as an important source of bioactive metabolites, among which phycobiliproteins (PBPs) are a class of water-soluble macromolecules of cyanobacteria with a wide range of applications. Massive proliferation of cyanobacteria can lead to excessive surface water blooms, of which removal, as a management measure, should be prioritized. In this study, the utilization of wild cyanobacteria biomass ( Aphanizomenon flos-aquae ) for extraction of phycobiliproteins is reported. Extraction of phycobiliproteins by conventional methods, such as homogenization, freeze-thaw cycles, and solid-liquid extraction, were optimized prior to ultrasound-assisted extraction. Standardization of ultrasonication for different parameters, such as ultrasonication amplitude (38, 114, and 190 μm) and ultrasonication time (1, 5.5, and 10 min), was carried out using a central composite design and response surface methodology for each of the primary techniques. A substantial increase on the individual and total phycobiliprotein yields was observed after ultrasonic treatment. The highest total PBP yield (115.37 mg/g of dry weight) was observed with samples treated with a homogenizer (30 min, 30 °C, and 1 cycle) combined with ultrasound treatment (8.7 min at 179 μm). Moreover, in vitro antioxidant capacity was observed for the obtained extracts in the Folin-Ciocalteu and ABTS *   + assays. In addition, a cytotoxic effect against C6 glioma cells was observed for A. flos-aquae PBPs. Conclusively, wild cyanobacteria could be considered as an alternative feedstock for recovery of PBPs.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.

Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Å crystal structure of PC from cyanobacterium Nostoc sp. WR13 (Nst-PC) is reported. The P6 3 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αβ) 3 trimeric rings. The structure has been refined to R factor 11.5% (R free 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and β chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates showing special spectral characteristics were prepared from the partly dissociated products of phycobilisomes from Polysiphonia urceolata. The absorption difference spectra between the aggregates and phycoerythrins showing normal spectral characteristics show peaks at 583 nm. The fluorescence emission difference spectra between the phycoerythrin aggregates and normal phycoerythrins show peaks at 602 nm. The special spectral characteristics of the PE aggregates disappeared when the PE aggregates were dissociated in deionized water. The intact phycobilisomes show similar characteristics as phycoerythrin-phycocyanin aggregates. When the peak values at 583 nm in absorption difference spectra were compared with those at 615 nm, it can be found that the amount of the 583 nm chromophores in the phycoerythrin-phycocyanin aggregates and intact phycobilisomes is similar to the amount of PCB chromophores in phycocyanins. It can be concluded that the 583 nm chromophores are at the interface between phycoerythrins and phycocyanins in phycobilisomes and transfer the light energy absorbed by other chromophores in phycoerythrins to the PCB chromophores in phycocyanins. A rod linker polypeptide with molecular weight of 40 kDa was found in the phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates, and it is believed to play roles in the spectral red shift of the aggregates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Phycobiliproteins (PBPs) are the main component of light-harvesting complexes in cyanobacteria and red algae. In addition to their important role in photosynthesis, PBPs have many potential applications in foods, cosmetics, medical diagnosis and treatment of diseases. However, basic researches and technological innovations are urgently needed for exploring those potentials, such as structure and function, their biosynthesis as well as downstream purification. For medical use and application, mechanisms underlying their therapeutic effects must be elucidated. Focusing on these issues, this article gives a critical review on the current status on PBPs, including their structures and functions, preparation processes and applications. In addition, key technical challenges and possible solutions are prospected., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

The objective of the present study was to identify peptides, based on active components of the red algae seaweed Pyropia yezoensis, able to inhibit the generation of reactive oxygen species (ROS), which is associated with aging and oxidative activities. Phycobilin, specific to red algae, covalently binds with water‑soluble proteins. There are three types of pigment bound proteins, known as phycobiliproteins (PBPs): Phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). In the present study, PBPs reported previously to have antioxidant activities in P. yezoensis were identified and, based on these data, several peptides were synthesized (PBP 1‑13) and their inhibition of ROS generation was examined. The existence of PBPs of each type, PE, PC and APC, was established in P. yezoensis and all were analyzed. In addition, PBP 1‑2 and 7‑9 peptides from PE were synthesized and showed antioxidant activities in HepG2 cells. In HepG2 cells, treatment with PBP2 reduced hydrogen peroxide‑mediated oxidative stress and restored the expression of superoxide dismutase (SOD). Furthermore, phosphorylated nuclear factor erythroid‑derived 2‑like 2 (Nrf2) was elevated by PBP2 treatment. Overall, these results suggested that Nrf2-SOD pathways may be involved in the PBP2‑mediated antioxidant effects. Therefore, from the investigations of P. yezoensis, several candidate peptides were identified with promising antioxidant and, potentially, anti‑aging properties.

Phycobilisomes are light-harvesting protein complexes and are widely distributed in red algae and cyanobacteria. Each phycobilisome contains highly fluorescent protein components called phycobiliproteins. Based upon the distinct physiochemical properties, phycobiliproteins are classified as allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin. In the present study, we describe purification and structural characterization of a novel phycocyanin and phycoerythrin isolated from a marine red macroalga, Centroceras clavulatum. The absorbance and fluorescence studies indicated that the purified proteins belong to R-Phycocyanin (R-PC) and R-Phycoerythrin (R-PE). The single bands under native-polyacrylamide gel electrophoresis revealed the intact molecular weights of R-PC and R-PE as 110kDa and 250kDa. The polypeptide compositions of the two proteins were demonstrated by SDS-PAGE. The result showed that R-PC contains two bands at 17 and 21kDa and were identified as α and β subunits through mass spectrometry based proteomics experiments. SDS-PAGE of R-PE showed three distinct bands at 18, 19 and 35kDa and was subsequently identified as α, β and γ subunits. The near-complete amino acid sequences of α and β subunits of R-PC and R-PE were derived from mass spectrometric data combined with Mascot software and multiple de novo sequencing tools followed by homology search and manual validation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Determining the key features of high-efficiency photosynthetic energy transfer remains an ongoing task. Recently, there has been evidence for the role of vibronic coherence in linking donor and acceptor states to redistribute oscillator strength for enhanced energy transfer. To gain further insights into the interplay between vibronic wavepackets and energy-transfer dynamics, we systematically compare four structurally related phycobiliproteins from cryptophyte algae by broad-band pump-probe spectroscopy and extend a parametric model based on global analysis to include vibrational wavepacket characterization. The four phycobiliproteins isolated from cryptophyte algae are two "open" structures and two "closed" structures. The closed structures exhibit strong exciton coupling in the central dimer. The dominant energy-transfer pathway occurs on the subpicosecond timescale across the largest energy gap in each of the proteins, from central to peripheral chromophores. All proteins exhibit a strong 1585 cm -1 coherent oscillation whose relative amplitude, a measure of vibronic intensity borrowing from resonance between donor and acceptor states, scales with both energy-transfer rates and damping rates. Central exciton splitting may aid in bringing the vibronically linked donor and acceptor states into better resonance resulting in the observed doubled rate in the closed structures. Several excited-state vibrational wavepackets persist on timescales relevant to energy transfer, highlighting the importance of further investigation of the interplay between electronic coupling and nuclear degrees of freedom in studies on high-efficiency photosynthesis.

The mechanisms controlling excitation energy transport (EET) in light-harvesting complexes remain controversial. Following the observation of long-lived beats in 2D electronic spectroscopy of PC645, vibronic coherence, the delocalization of excited states between pigments supported by a resonant vibration, has been proposed to enable direct excitation transport from the highest-energy to the lowest-energy pigments, bypassing a collection of intermediate states. Here, we instead show that for phycobiliprotein PC645 an incoherent vibronic transport mechanism is at play. We quantify the solvation dynamics of individual pigments using ab initio quantum mechanics/molecular mechanics (QM/MM) nuclear dynamics. Our atomistic spectral densities reproduce experimental observations ranging from absorption and fluorescence spectra to the timescales and selectivity of down-conversion observed in transient absorption measurements. We construct a general model for vibronic dimers and establish the parameter regimes of coherent and incoherent vibronic transport. We demonstrate that direct down-conversion in PC645 proceeds incoherently, enhanced by large reorganization energies and a broad collection of high-frequency vibrations. We suggest that a similar incoherent mechanism is appropriate across phycobiliproteins and represents a potential design principle for nanoscale control of EET., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-α-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette Cys in vitro A blue-shifted tripyrrole formed from maquette-bound phycocyanobilin displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in Escherichia coli to improve the efficiency of bilin binding and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices., (© 2018 The Author(s).)

Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α 1 β·α 2 β-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.

The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin β-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The cyanobacterium Acaryochloris marina developed two types of antenna complexes, which contain chlorophyll-d (Chl d) and phycocyanobilin (PCB) as light-harvesting pigment molecules, respectively. The latter membrane-extrinsic complexes are denoted as phycobiliproteins (PBPs). Spectral hole burning was employed to study excitation energy transfer and electron-phonon coupling in PBPs. The data reveal a rich spectral substructure with a total of four low-energy electronic states whose absorption bands peak at 633, 644, 654, and at about 673 nm. The electronic states at ~633 and 644 nm can be tentatively attributed to phycocyanin (PC) and allophycocyanin (APC), respectively. The remaining low-energy electronic states including the terminal emitter at 673 nm may be associated with different isoforms of PC, APC, or the linker protein. Furthermore, the hole burning data reveal a large number of excited state vibrational frequencies, which are characteristic for the chromophore PCB. In summary, the results are in good agreement with the low-energy level structure of PBPs and electron-phonon coupling parameters reported by Gryliuk et al. (BBA 1837:1490-1499, 2014) based on difference fluorescence line-narrowing experiments.

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 structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆T m of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αβ) 3 trimer and (αβ) 6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and β subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Auxiliary metabolic genes (AMG) are commonly found in the genomes of phages that infect cyanobacteria and increase the fitness of the cyanophage. AMGs are often homologs of host genes, and also typically related to photosynthesis. For example, the Φ cpeT gene in the cyanophage P-HM1 encodes a putative phycobiliprotein lyase related to cyanobacterial T-type lyases, which facilitate attachment of linear tetrapyrrole chromophores to Cys-155 of phycobiliprotein β-subunits, suggesting that ΦCpeT may also help assemble light-harvesting phycobiliproteins during infection. To investigate this possibility, we structurally and biochemically characterized recombinant ΦCpeT. The solved crystal structure of ΦCpeT at 1.8-Å resolution revealed that the protein adopts a similar fold as the cyanobacterial T-type lyase CpcT from Nostoc sp. PCC7120 but overall is more compact and smaller. ΦCpeT specifically binds phycoerythrobilin (PEB) in vitro leading to a tight complex that can also be formed in Escherichia coli when it is co-expressed with genes encoding PEB biosynthesis ( i.e. ho1 and pebS ). The formed ΦCpeT·PEB complex was very stable as the chromophore was not lost during chromatography and displayed a strong red fluorescence with a fluorescence quantum yield of Φ F = 0.3. This complex was not directly able to transfer PEB to the host phycobiliprotein β-subunit. However, it could assist the host lyase CpeS in its function by providing a pool of readily available PEB, a feature that might be important for fast phycobiliprotein assembly during phage infection., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In cyanobacteria, phycobiliproteins (PBS) show excellent energy transfer among the chromophores absorbing over most of the visible. The energy transfers are used to study phycobilisome assembly and bioimaging. Using All4261GAF2(C81L) as energy donor, ApcE(1-240/Δ87-130) as energy acceptor, we co-expressed fusion protein ApcE(1-240/Δ87-130)::All4261GAF2(C81L) with phycobiliprotein in Escherichia Coli and studied the energy transfer between two protein domains. With N-terminal His6 tag, ApcE(1-240/Δ87-130)::All4261GAF2(C81L) cannot be purified by nickel-affinity column. We added six histidines in the C-terminal of ApcE(1-240/Δ87-130)::All4261GAF2(C81L) and co-expressed it with phycobiliprotein. ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was purified successfully and only singly chromophorylated at All4261GAF2(C81L)His6 domain. The singly chromophorylate ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was incubated with fresh PCB and the doubly chromophorylated PCB-ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was obtained. The double chromophored fusion protein absorbed light in the range of 615-660 nm, and fluoresced only at 668 nm. Photochemistry analysis showed that excitation energy transfer from the short-wavelength absorbing at All4261GAF2(C81L) domain was achieved successfully to the long-wavelength absorbing at the ApcE(1-240/Δ87-130) domain., (Copyright © 2016. Published by Elsevier Inc.)

Algae can tolerate a broad range of growing conditions but extreme conditions may lead to the generation of highly dangerous reactive oxygen species (ROS), which may cause the deterioration of cell metabolism and damage cellular components. The antioxidants produced by algae alleviate the harmful effects of ROS. While the enhancement of antioxidant production in blue green algae under stress has been reported, the antioxidant response to changes in pH levels requires further investigation. This study presents the effect of pH changes on the antioxidant activity and productivity of the blue green alga Spirulina (Arthrospira) platensis. The algal dry weight (DW) was greatly enhanced at pH 9.0. The highest content of chlorophyll a and carotenoids (10.6 and 2.4mg/g DW, respectively) was recorded at pH 8.5. The highest phenolic content (12.1mg gallic acid equivalent (GAE)/g DW) was recorded at pH 9.5. The maximum production of total phycobiliprotein (159mg/g DW) was obtained at pH 9.0. The antioxidant activities of radical scavenging activity, reducing power and chelating activity were highest at pH 9.0 with an increase of 567, 250 and 206% compared to the positive control, respectively. Variation in the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was also reported. While the high alkaline pH may favor the overproduction of antioxidants, normal cell metabolism and membrane function is unaffected, as shown by growth and chlorophyll content, which suggests that these conditions are suitable for further studies on the harvest of antioxidants from S. platensis., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)