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Molecules (Basel, Switzerland) Mar 2016A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained...
Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314.
A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB) producing genes (hoxI and pcyA), while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB) and the lyase gene (cpcU, cpcS, or cpcU/S) were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.
Topics: Amino Acid Sequence; Bile Pigments; Cloning, Molecular; Cyanobacteria; Gene Expression; Lyases; Phycobilins; Phycocyanin; Phylogeny; Protein Binding; Protein Multimerization; Recombinant Proteins
PubMed: 26999083
DOI: 10.3390/molecules21030357 -
Proceedings of the National Academy of... Mar 2016In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS),...
In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5-10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.
Topics: Bacterial Proteins; Cross-Linking Reagents; Energy Transfer; Glutaral; Models, Chemical; Models, Molecular; Mutation; Phycobilins; Phycobilisomes; Phycocyanin; Protein Conformation; Protein Subunits; Radiation Tolerance; Spectrometry, Fluorescence; Synechocystis; Tandem Mass Spectrometry
PubMed: 26957606
DOI: 10.1073/pnas.1523680113 -
PloS One 2016As a consequence of the inhibition of one of the steps in the biosynthesis of the photopigments chlorophyll and phycobilin, the red microalga Galdieria partita excretes...
As a consequence of the inhibition of one of the steps in the biosynthesis of the photopigments chlorophyll and phycobilin, the red microalga Galdieria partita excretes coproporphyrinogen III in the medium when growing on glucose. No coproporphyrinogen III was found when the closely related red microalgae G. sulphuraria strain 074G was grown on glucose and excessive amounts of oxygen. When under the same conditions oxygen was limiting, coproporphyrinogen III was present in the medium. We conclude that not glucose but the amount of oxygen in the medium results in the accumulation of coproporphyrinogen III. This is explained by the inactivition of the oxygen-dependent coproporphyrinogen III oxidase that converts coproporhyrinogen III to protoporphyrinogen IX, one of the intermediate steps in the biosynthesis of chlorophyl and phycobilin.
Topics: Dose-Response Relationship, Drug; Microalgae; Oxygen; Pigments, Biological; Porphyrins; Rhodophyta
PubMed: 26859750
DOI: 10.1371/journal.pone.0148358 -
Scientific Reports Dec 2015Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed....
Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Biliverdine; HeLa Cells; Humans; Luminescent Proteins; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Mutagenesis; Phycobilins; Phycocyanin; Protein Structure, Tertiary; Rhodopseudomonas; Sequence Alignment; Spectrometry, Fluorescence; Spectroscopy, Near-Infrared
PubMed: 26679720
DOI: 10.1038/srep18348 -
Acta Crystallographica. Section F,... Aug 2015The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE...
The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, β = 82.50, γ = 60.34°. The molecular-replacement solution confirmed the presence of 12 αβ monomers in the P1 cell. The Phormidium PE elutes as an (αβ)3 trimer of αβ monomers from a molecular-sieve column and exists as [(αβ)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher-order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.
Topics: Amino Acid Sequence; Ammonium Sulfate; Crystallization; Crystallography, X-Ray; Cyanobacteria; Gene Expression; Models, Molecular; Molecular Sequence Data; Phycobilins; Phycoerythrin; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Sequence Alignment; Structural Homology, Protein
PubMed: 26249689
DOI: 10.1107/S2053230X15010134 -
World Journal of Gastroenterology May 2015To investigate the hepatoprotective effects of phycocyanobilin (PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride...
AIM
To investigate the hepatoprotective effects of phycocyanobilin (PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride (CCl4) treatment.
METHODS
C57BL/6 mice were orally administered PCB 100 mg/kg for 4 d after CCl4 injection, and then the serum and liver tissue of the mice were collected at days 1, 2, 3, 5 and 7 after CCl4 treatment. A series of evaluations were performed to identify the curative effects on liver injury and recovery. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and superoxide dismutase (SOD) were detected to indirectly assess the anti-inflammatory effects of PCB. Meanwhile, we detected the expressions of hepatocyte growth factor, transforming growth factor alpha (TGF-α), TGF-β, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), the factors which are associated with inflammation and liver regeneration. The protein expressions of proliferating cell nuclear antigen (PCNA), TNF-α and cytochrome C were detected by western blot. Furthermore, the survival rates were analyzed of mice which were administered a lethal dose of CCl4 (2.6 mg/kg) with or without PCB.
RESULTS
In our research, PCB showed a strongly anti-inflammatory effect on CCl4-induced liver injury in mice. The ALT was significantly decreased after CCl4 treatment from day 1 (P < 0.01) and the AST was significantly decreased from day 2 (P < 0.001). Both albumin and liver SOD were increased from day 2 (P < 0.001 and P < 0.01), but serum SOD levels did not show a significant increase (P > 0.05). PCB protected the structure of liver from the injury by CCl4. TUNEL assay showed that PCB dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control (101.0 ± 25.4 vs 25.7 ± 6.4, P < 0.01). The result of western blotting showed that PCB could increase PCNA expression, decrease TNF-α and cytochrome C expression. Furthermore, data shows that PCB could improve the survival rate of acute liver failure (ALF) mice which were injected with a lethal dose of CCl4 (60.0% vs 20.0%).
CONCLUSION
Our study indicated that PCB could be an ideal candidate for reversing acute liver injury or ALF.
Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Biomarkers; Carbon Tetrachloride; Cell Proliferation; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Liver; Liver Failure, Acute; Liver Regeneration; Male; Mice, Inbred C57BL; Necrosis; Phycobilins; Phycocyanin; Time Factors
PubMed: 25987768
DOI: 10.3748/wjg.v21.i18.5465 -
Photosynthesis Research Jan 2016Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method...
Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.
Topics: Computer Simulation; Fluorescence; Fluorometry; Models, Biological; Mutation; Photosystem I Protein Complex; Photosystem II Protein Complex; Phycobilisomes; Phycocyanin; Protein Subunits; Synechocystis; Time Factors
PubMed: 25893897
DOI: 10.1007/s11120-015-0141-x -
Missouri Medicine 2015Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive...
Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive pulmonary disease observed in smokers. Nicotine, via sympathetic stimulation, increases risk for both vascular disease and cancer. Comprehensive suppression of sympathetic activity with the well-tolerated drug carvedilol, which inhibits betal 1, beta2 and alphal adrenergic receptors, may be protective to smokers and other nicotine addicts. The soluble aldehydes and ketones in tobacco smoke appear to exert their adverse effects through activation of NADPH oxidase complexes in vascular tissues and in the lungs. The phytochemical phycocyanobilin (PhyCB), richly supplied by the edible cyanobacterium spirulina, in studies on rodents and in human cell cultures has shown the ability to safely mimic intracellular bilirubin's physiological role as an inhibitor ofNADPH oxidase activity. It therefore may have potential for mitigating the pro-oxidative effects of tobacco smoke aldehydes and ketones. Joint administration of carvedilol and spirulina merits exploration as a strategy for moderating the pathogenic impact of smoking in chronic tobacco users who either fail to quit or refuse to try cessation of tobacco. Carvedilol may be appropriate for those who manage a nicotine addiction in other ways (smokeless tobacco, e-cigarettes, nicotine gum). Further clinical studies to evaluate the impact of carvedilol on cardiovascular risk factors in nicotine addicts, and rodent studies to assess markers of lung inflammation in smoke- exposed rodents fed PhyCB, are recommended.
Topics: Adrenergic beta-Antagonists; Carbazoles; Cardiovascular Diseases; Carvedilol; Humans; Neoplasms; Phycobilins; Phycocyanin; Propanolamines; Smoking Cessation; Spirulina; Tobacco Use Disorder
PubMed: 25812281
DOI: No ID Found -
Scientific Reports Jan 2015Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors in cyanobacteria that absorb visible and near-ultraviolet light. CBCRs are divided into two...
Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors in cyanobacteria that absorb visible and near-ultraviolet light. CBCRs are divided into two types based on the type of chromophore they contain: phycocyanobilin (PCB) or phycoviolobilin (PVB). PCB-binding CBCRs reversibly photoconvert at relatively long wavelengths, i.e., the blue-to-red region, whereas PVB-binding CBCRs reversibly photoconvert at shorter wavelengths, i.e., the near-ultraviolet to green region. Notably, prior to this report, CBCRs containing biliverdin (BV), which absorbs at longer wavelengths than do PCB and PVB, have not been found. Herein, we report that the typical red/green CBCR AM1_1557 from the chlorophyll d-bearing cyanobacterium Acaryochloris marina can bind BV almost comparable to PCB. This BV-bound holoprotein reversibly photoconverts between a far red light-absorbing form (Pfr, λmax = 697 nm) and an orange light-absorbing form (Po, λmax = 622 nm). At room temperature, Pfr fluoresces with a maximum at 730 nm. These spectral features are red-shifted by 48~77 nm compared with those of the PCB-bound domain. Because the absorbance of chlorophyll d is red-shifted compared with that of chlorophyll a, the BV-bound AM1_1557 may be a physiologically relevant feature of A. marina and is potentially useful as an optogenetic switch and/or fluorescence imager.
Topics: Biliverdine; Chlorophyll; Cyanobacteria; Light; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Binding
PubMed: 25609645
DOI: 10.1038/srep07950 -
Biophysical Journal Nov 2014Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These...
Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 ?s. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.
Topics: Cyanobacteria; Light; Molecular Structure; Photochemical Processes; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Conformation; Spectrum Analysis
PubMed: 25418104
DOI: 10.1016/j.bpj.2014.09.020