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Biochimica Et Biophysica Acta.... Apr 2019The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed...
The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.
Topics: Bacterial Proteins; Carotenoids; Phycobilisomes; Spectrometry, Fluorescence; Synechocystis
PubMed: 30721662
DOI: 10.1016/j.bbabio.2019.01.007 -
Biochimica Et Biophysica Acta.... Apr 2019In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of...
In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77 K. The study demonstrates that if complexes are stabilized by high phosphate (900 mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50 mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.
Topics: Bacterial Proteins; Cyanobacteria; Phycobilins; Phycobilisomes; Phycocyanin; Spectrometry, Fluorescence
PubMed: 30703363
DOI: 10.1016/j.bbabio.2019.01.002 -
The Journal of Biological Chemistry Mar 2019Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due...
Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, , in the cyanobacterium Analyzing the phenotype of the deletion, we found that is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes.
Topics: Cyanobacteria; Phycobilins; Phycoerythrin; Recombinant Proteins
PubMed: 30670589
DOI: 10.1074/jbc.RA118.007221 -
The Journal of Biological Chemistry Nov 2018Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible...
Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 and , which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Bacterial Proteins; Carrier Proteins; Histidine Kinase; Kinetics; Molecular Docking Simulation; Mutation; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Binding; Protein Conformation; Sequence Alignment; Synechocystis
PubMed: 30242127
DOI: 10.1074/jbc.RA118.003830 -
Physical Chemistry Chemical Physics :... Aug 2018The light-harvesting mechanisms of cryptophyte antenna complexes have attracted considerable attention due to their ability to exhibit maximal photosynthetic activity...
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.
Topics: Cryptophyta; Hydrogen-Ion Concentration; Light; Models, Chemical; Molecular Dynamics Simulation; Phycobilins; Phycobiliproteins; Protons; Quantum Theory; Thermodynamics
PubMed: 30105318
DOI: 10.1039/c8cp02541j -
Nature Communications May 2018The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned...
The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.
Topics: Clone Cells; Gene Editing; Gene Expression; Genes, Synthetic; Genetic Engineering; Genome, Fungal; Integrases; Light; Phycobilins; Phycocyanin; Plasmids; Recombination, Genetic; Saccharomyces cerevisiae; Selection, Genetic
PubMed: 29789561
DOI: 10.1038/s41467-017-02208-6 -
International Journal of Molecular... Aug 2018The 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...
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.
Topics: Antioxidants; Hep G2 Cells; Humans; Oxidative Stress; Peptides; Phycobiliproteins; Reactive Oxygen Species; Rhodophyta; Superoxide Dismutase
PubMed: 29717771
DOI: 10.3892/ijmm.2018.3650 -
PloS One 2018Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to...
Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αβ)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and β R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the β subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.
Topics: Amino Acid Sequence; Gracilaria; Phycobilins; Phycoerythrin; Plant Proteins; Protein Subunits; Sequence Analysis; Urobilin
PubMed: 29634783
DOI: 10.1371/journal.pone.0195656 -
Scientific Reports Dec 2017Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical...
Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.
Topics: Bacterial Proteins; Light-Harvesting Protein Complexes; Photosynthesis; Phycobilins; Phycocyanin; Phycoerythrin; Stochastic Processes; Synechocystis
PubMed: 29196704
DOI: 10.1038/s41598-017-16834-z -
Proceedings of the National Academy of... Dec 2017The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally...
The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally distinct lyase families known. The heterodimeric E/F type is specific for attaching bilins covalently to α-subunits of phycocyanins and phycoerythrins. Unlike other lyases, the lyase also has chromophore-detaching activity. A subclass of the E/F-type lyases is, furthermore, capable of chemically modifying the chromophore. Although these enzymes were characterized >25 y ago, their structures remained unknown. We determined the crystal structure of the heterodimer of CpcE/F from sp. PCC7120 at 1.89-Å resolution. Both subunits are twisted, crescent-shaped α-solenoid structures. CpcE has 15 and CpcF 10 helices. The inner (concave) layer of CpcE (helices h2, 4, 6, 8, 10, 12, and 14) and the outer (convex) layer of CpcF (h16, 18, 20, 22, and 24) form a cavity into which the phycocyanobilin chromophore can be modeled. This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity. The structure of a structurally related, isomerizing lyase, PecE/F, that converts phycocyanobilin into phycoviolobilin, was modeled using the CpcE/F structure as template. A HC motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21, supporting the proposal that the nucleophilic addition of Cys-88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin. Also, the structure of NblB, involved in phycobilisome degradation could be modeled using CpcE as template. Combined with CpcF, NblB shows a low chromophore-detaching activity.
Topics: Bacterial Proteins; Lyases; Molecular Dynamics Simulation; Nostoc; Phycobilins; Phycocyanin; Protein Domains
PubMed: 29180420
DOI: 10.1073/pnas.1715495114