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Molecules (Basel, Switzerland) Nov 2020We present a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics-statistical approach for the interpretation of nuclear magnetic resonance (NMR)...
We present a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics-statistical approach for the interpretation of nuclear magnetic resonance (NMR) chemical shift patterns in phycocyanobilin (PCB). These were originally associated with colour tuning upon photoproduct formation in red/green-absorbing cyanobacteriochrome AnPixJg2 and red/far-red-absorbing phytochrome Cph1Δ2. We pursue an indirect approach without computation of the absorption frequencies since the molecular geometry of cofactor and protein are not accurately known. Instead, we resort to a heuristic determination of the conjugation length in PCB through the experimental NMR chemical shift patterns, supported by quantum chemical calculations. We have found a characteristic correlation pattern of 13C chemical shifts to specific bond orders within the π-conjugated system, which rests on the relative position of carbon atoms with respect to electron-withdrawing groups and the polarisation of covalent bonds. We propose the inversion of this regioselective relationship using multivariate statistics and to apply it to the known experimental NMR chemical shifts in order to predict changes in the bond alternation pattern. Therefrom the extent of electronic conjugation, and eventually the change in absorption frequency, can be derived. In the process, the consultation of explicit mesomeric formulae plays an important role to qualitatively account for possible conjugation scenarios of the chromophore. While we are able to consistently associate the NMR chemical shifts with hypsochromic and bathochromic shifts in the Pg and Pfr, our approach represents an alternative method to increase the explanatory power of NMR spectroscopic data in proteins.
Topics: Carbon; Carbon Isotopes; Color; Magnetic Resonance Spectroscopy; Models, Theoretical; Molecular Conformation; Molecular Dynamics Simulation; Phycobilins; Phycocyanin
PubMed: 33255423
DOI: 10.3390/molecules25235505 -
Journal of Microbiology and... Feb 2021Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF...
Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from , covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λ = 520 nm, λ = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.
Topics: Bacterial Proteins; Copper; Fluorescence; Light; Luminescent Proteins; Phycobilins; Phycocyanin; Phycoerythrin; Phytochrome; Protein Domains; Spirulina
PubMed: 33203817
DOI: 10.4014/jmb.2009.09048 -
Nature Ecology & Evolution Jan 2021Stretching and bending vibrations of water molecules absorb photons of specific wavelengths, a phenomenon that constrains light energy available for aquatic...
Stretching and bending vibrations of water molecules absorb photons of specific wavelengths, a phenomenon that constrains light energy available for aquatic photosynthesis. Previous work suggested that these absorption properties of water create a series of spectral niches but the theory was still too simplified to enable prediction of the spectral niches in real aquatic ecosystems. Here, we show with a state-of-the-art radiative transfer model that the vibrational modes of the water molecule delineate five spectral niches, in the violet, blue, green, orange and red parts of the spectrum. These five niches are effectively captured by chlorophylls and phycobilin pigments of cyanobacteria and their eukaryotic descendants. Global distributions of the spectral niches are predicted by satellite remote sensing and validated with observed large-scale distribution patterns of cyanobacterial pigment types. Our findings provide an elegant explanation for the biogeographical distributions of photosynthetic pigments across the lakes and oceans of our planet.
Topics: Ecosystem; Lakes; Oceans and Seas; Photosynthesis; Vibration; Water
PubMed: 33168993
DOI: 10.1038/s41559-020-01330-x -
ACS Chemical Biology Nov 2020Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate...
Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, , , , and , were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.
Topics: Biosynthetic Pathways; Cell Line; Genes, Bacterial; HeLa Cells; Humans; Optogenetics; Phycobilins; Phycocyanin; Synechocystis; Thermosynechococcus
PubMed: 33164485
DOI: 10.1021/acschembio.0c00477 -
The Journal of Biological Chemistry 2021Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to...
Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.
Topics: Amino Acid Sequence; Bacterial Proteins; Chromatography, Liquid; Isomerases; Marine Biology; Phycobilins; Phycoerythrin; Phylogeny; Recombinant Proteins; Synechococcus; Tandem Mass Spectrometry; Urobilin
PubMed: 33154169
DOI: 10.1074/jbc.RA120.015289 -
Proceedings of the National Academy of... Nov 2020Cyanobacteriochromes (CBCRs) are photoswitchable linear tetrapyrrole (bilin)-based light sensors in the phytochrome superfamily with a broad spectral range from the near...
Cyanobacteriochromes (CBCRs) are photoswitchable linear tetrapyrrole (bilin)-based light sensors in the phytochrome superfamily with a broad spectral range from the near UV through the far red (330 to 760 nm). The recent discovery of far-red absorbing CBCRs (frCBCRs) has garnered considerable interest from the optogenetic and imaging communities because of the deep penetrance of far-red light into mammalian tissue and the small size of the CBCR protein scaffold. The present studies were undertaken to determine the structural basis for far-red absorption by JSC1_58120g3, a frCBCR from the thermophilic cyanobacterium sp. JSC-1 that is a representative member of a phylogenetically distinct class. Unlike most CBCRs that bind phycocyanobilin (PCB), a phycobilin naturally occurring in cyanobacteria and only a few eukaryotic phototrophs, JSC1_58120g3's far-red absorption arises from incorporation of the PCB biosynthetic intermediate 18,18-dihydrobiliverdin (18,18-DHBV) rather than the more reduced and more abundant PCB. JSC1_58120g3 can also yield a far-red-absorbing adduct with the more widespread linear tetrapyrrole biliverdin IXα (BV), thus circumventing the need to coproduce or supplement optogenetic cell lines with PCB. Using high-resolution X-ray crystal structures of 18,18-DHBV and BV adducts of JSC1_58120g3 along with structure-guided mutagenesis, we have defined residues critical for its verdin-binding preference and far-red absorption. Far-red sensing and verdin incorporation make this frCBCR lineage an attractive template for developing robust optogenetic and imaging reagents for deep tissue applications.
Topics: Bacterial Proteins; Biliverdine; Cyanobacteria; Light; Photoreceptor Cells; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Phytochrome; Porphyrins
PubMed: 33106421
DOI: 10.1073/pnas.2016047117 -
AMB Express Sep 2020In the last years, the acidothermophilic red microalga Galdieria sulphuraria has been increasingly studied for industrial applications such as wastewater treatment,...
In the last years, the acidothermophilic red microalga Galdieria sulphuraria has been increasingly studied for industrial applications such as wastewater treatment, recovery of rare earth elements, production of phycobilins. However, even now it is not possible an industrial cultivation of this organism because biotechnological research on G. sulphuraria and allied species is relatively recent and fragmented. Having in mind a possible scale-up for commercial applications, we have compared the growth and photosynthetic performance of G. sulphuraria in four suspended systems (Inclined bubble column, Decanter Laboratory Flask, Tubular Bioreactor, Ultra-flat plate bioreactor) and one immobilized system (Twin Layer Sytem). The results showed that G. sulphuraria had the highest growth, productivity and photosynthetic performance, when grown on the immobilized system, which also offers some economics advantages.
PubMed: 32955638
DOI: 10.1186/s13568-020-01110-7 -
Cells Sep 2020It is estimated that the genus is responsible for about 17% of net primary production in the Global Ocean. Blooms of these organisms are observed in tropical,...
It is estimated that the genus is responsible for about 17% of net primary production in the Global Ocean. Blooms of these organisms are observed in tropical, subtropical and even temperate zones, and they have been recorded recently even beyond the polar circle. The long-term scenarios forecast a growing expansion of sp. and its area of dominance. This is, among others, due to their high physiological plasticity in relation to changing environmental conditions. Three phenotypes of the genus sp. (Type 1, Type 2, and Type 3a) were tested in controlled laboratory conditions in order to identify their response to various irradiance (10, 55, 100 and 145 µmol photons m s) and temperature (15, 22.5 and 30 °C) conditions. The highest total pigment content per cell was recorded at 10 μmol photons m s at all temperature variants with the clear dominance of phycobilins among all the pigments. In almost every variant the highest growth rate was recorded for the Type 1. The lowest growth rates were observed, in general, for the Type 3a. However, it was recognized to be less temperature sensitive in comparison to the other two types and rather light-driven with the highest plasticity and adaptation potential. The highest amounts of carotenoids were produced by Type 2 which also showed signs of the cell stress even around 55 μmol photons m s at 15 °C and 22.5 °C. This may imply that the Type 2 is the most susceptible to higher irradiances. Picocyanobacteria sp. require less light intensity to achieve the maximum rate of photosynthesis than larger algae. They also tolerate a wide range of temperatures which combined together make them gain a powerful competitive advantage. Our results will provide key information for the ecohydrodynamical model development. Thus, this work would be an important link in forecasting future changes in the occurrence of these organisms in the context of global warming.
Topics: Humans; Phenotype; Photosynthesis; Synechococcus; Temperature
PubMed: 32899279
DOI: 10.3390/cells9092030 -
The Plant Journal : For Cell and... Nov 2020The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the...
The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the covalent attachment of chromophores to the antenna proteins. The Arabidopsis CRUMPLED LEAF (CRL) protein is a homolog of the cyanobacterial CpcT lyase. Loss of CRL leads to multiple lesions, including localized foliar cell death, constitutive expression of stress-related nuclear genes, abnormal cell cycle, and impaired plastid division. Notwithstanding the apparent phenotypes, the function of CRL still remains elusive. To gain insight into the function of CRL, we examined whether CRL still retains the capacity to bind with the bacterial chromophore phycocyanobilin (PCB) and its plant analog phytochromobilin (PΦB). The revealed structure of the CpcT domain of CRL is comparable to that of the CpcT lyase, despite the low sequence identity. The subsequent in vitro biochemical assays found, as shown for the CpcT lyase, that PCB/PΦB binds to the CRL dimer. However, some mutant forms of CRL, substantially compromised in their bilin-binding ability, still restore the crl-induced multiple lesions. These results suggest that although CRL retains the bilin-binding pocket, it seems not functionally associated with the crl-induced multiple lesions.
Topics: Arabidopsis; Arabidopsis Proteins; Bile Pigments; Cell Division; Cyanobacteria; Lyases; Mutation; Phenotype; Phycobilins; Phycobiliproteins; Phycobilisomes; Phycocyanin; Plastids; Protein Binding
PubMed: 32860438
DOI: 10.1111/tpj.14974 -
Biochimica Et Biophysica Acta.... Dec 2020Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae....
Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the β-subunit of PE. MS analyses of the recombinant β-subunit of PE coexpressed with CpeT and CpeZ show that PEB is attached at Cys-165. Purified phycobilisomes from a cpeT knockout mutant and wild type (WT) samples from F. diplosiphon were analyzed and compared. The cpeT mutant contained much less PE and more phycocyanin than WT cells grown under green light, conditions which should maximize the production of PE. In addition, Northern blot analyses showed that the cpeCDESTR operon mRNAs were upregulated while the cpeBcpeA mRNAs were downregulated in the cpeT mutant strain when compared with WT, suggesting that CpeT may also play a direct or indirect regulatory role in transcription of these operons or their mRNA stability, in addition to its role as a PEB lyase for Cys-165 on β-PE.
Topics: Amino Acid Sequence; Bacterial Proteins; Cyanobacteria; Cysteine; Gene Deletion; Genes, Bacterial; Lyases; Molecular Chaperones; Mutant Proteins; Operon; Peptides; Phenotype; Phycobilins; Phycoerythrin; Recombinant Proteins
PubMed: 32777305
DOI: 10.1016/j.bbabio.2020.148284