RESUMO
Phycobiliproteins are a class of light-harvesting fluorescent proteins existing in cyanobacteria and microalgae, which harvest light and convert it into electricity. Owing to recent demands on environmental-friendly and renewable apparatuses, phycobiliproteins have attracted substantial interest in bioenergy and sustainable devices. However, converting energy from biological materials remains challenging to date. Herein, we report a novel scheme to enhance biological light-harvesting through light-matter interactions at the biointerface of whispering-gallery modes (WGMs), where phycobiliproteins were employed as the active gain material. By exploiting microdroplets as a carrier for light-harvesting biomaterials, strong local electric field enhancement and photon confinement at the cavity interface resulted in significantly enhanced bio-photoelectricity. A threshold-like behavior was discovered in photocurrent enhancement and the WGM modulated fluorescence. Systematic studies of biologically produced photoelectricity and optical mode resonance were carried out to illustrate the impact of the cavity quality factor, structural geometry, and refractive indices. Finally, a biomimetic system was investigated by exploiting cascade energy transfer in phycobiliprotein assembly composed of three light-harvesting proteins. The key findings not only highlight the critical role of optical cavity in light-harvesting but also offer deep insights into light energy coupling in biomaterials.
Assuntos
Materiais Biomiméticos/química , Ficocianina/química , Ficoeritrina/química , Materiais Biomiméticos/efeitos da radiação , Eletricidade , Luz , Cristais Líquidos/química , Cristais Líquidos/efeitos da radiação , Óptica e Fotônica , Ficocianina/efeitos da radiação , Ficoeritrina/efeitos da radiação , Estudo de Prova de Conceito , RefratometriaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Ficobilissomas/química , Synechocystis/metabolismo , Proteínas de Bactérias/fisiologia , Reagentes de Ligações Cruzadas/farmacologia , Transferência de Energia , Glutaral/farmacologia , Modelos Químicos , Modelos Moleculares , Mutação , Ficobilinas/efeitos da radiação , Ficobilissomas/metabolismo , Ficobilissomas/efeitos da radiação , Ficocianina/genética , Ficocianina/metabolismo , Ficocianina/efeitos da radiação , Conformação Proteica/efeitos da radiação , Subunidades Proteicas , Tolerância a Radiação , Espectrometria de Fluorescência , Synechocystis/genética , Synechocystis/efeitos da radiação , Espectrometria de Massas em TandemRESUMO
Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹5N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.
Assuntos
Proteínas de Bactérias/química , Modelos Moleculares , Nostoc/metabolismo , Ficobilinas/química , Ficocianina/química , Pigmentos Biológicos/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Sítios de Ligação , Isótopos de Carbono , Interações Hidrofóbicas e Hidrofílicas , Imageamento Tridimensional , Marcação por Isótopo , Luz , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/efeitos da radiação , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Ficobilinas/metabolismo , Ficobilinas/efeitos da radiação , Ficocianina/metabolismo , Ficocianina/efeitos da radiação , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Pigmentos Biológicos/efeitos da radiação , Conformação Proteica/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , EstereoisomerismoRESUMO
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.
Assuntos
Cianobactérias/efeitos da radiação , Luz , Fotorreceptores Microbianos/efeitos da radiação , Ficobilinas/efeitos da radiação , Cianobactérias/química , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Ficobilinas/química , Ficocianina/química , Ficocianina/efeitos da radiação , Conformação Proteica/efeitos da radiação , Análise EspectralRESUMO
A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos da radiação , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criptocromos/biossíntese , Criptocromos/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Galactoquinase/genética , Genes Reporter , Ficobilinas/farmacologia , Ficobilinas/efeitos da radiação , Ficocianina/farmacologia , Ficocianina/efeitos da radiação , Fitocromo B/biossíntese , Fitocromo B/genética , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The geometric relaxation following light absorption of the biliverdin, phycocyanobilin and phytochromobilin tetrapyrrole chromophores of bacterial, cyanobacterial and plant phytochromes has been investigated using density functional theory methods. Considering stereoisomers relevant for both red-absorbing Pr and far-red-absorbing Pfr forms of the photoreceptor, it is found that the initial excited-state evolution is dominated by torsional motion at the C10-C11 bond. This holds true for all three chromophores and irrespective of which configuration the chromophores adopt. This finding suggests that the photochromic cycling of phytochromes between their Pr and Pfr forms, which is known to be governed by Z/E photoisomerizations at the C15-C16 bond, relies on interactions between the chromophore and the protein to prevent photoisomerizations at C10-C11. Further, it is found that the uneven distribution of positive charge between the pyrrole rings is a major factor for the photochemical reactivity of the C10-C11 bond.
Assuntos
Pigmentos Biliares/química , Fitocromo/química , Pigmentos Biliares/efeitos da radiação , Biliverdina/análogos & derivados , Biliverdina/química , Biliverdina/efeitos da radiação , Modelos Moleculares , Ficobilinas/química , Ficobilinas/efeitos da radiação , Ficocianina/química , Ficocianina/efeitos da radiação , Teoria Quântica , Estereoisomerismo , TermodinâmicaRESUMO
In the present study, we investigated the mechanism of UV-B radiation induced damage to the light harvesting apparatus of the cyanobacterium Synechocystis 6803. Liquid chromatography analysis and spectroscopy investigations performed on phycobilisomes or isolated biliproteins irradiated with moderate UV-B intensity (1.3 W/m(2)) revealed rapid destruction of beta-phycocyanin and a slower damage of the other biliproteins, alpha-phycocyanin and both alpha and beta-allophycocyanin. EPR spin trapping measurements revealed that carbon centered adducts of the spin trap DMPO were formed. This evidence indicates that free radicals produced from bilins probably attack the polypeptide chain of protein inducing its degradation. Our results show that the bilin chromophore is the main target of UV-B irradiation, causing structural changes, which in turn induce reaction of the chromophore with atmospheric oxygen and lead to production of reactive radicals. Our results also demonstrate that beta-phycocyanin is the most affected biliprotein, probably due to the presence of two bilins as chromophore.
Assuntos
Proteínas de Bactérias/efeitos da radiação , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Synechocystis/efeitos da radiação , Raios Ultravioleta , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância de Spin Eletrônica , Ficobilissomas/efeitos da radiação , Ficocianina/efeitos da radiação , EspectrofotometriaRESUMO
Phycoerythrocyanin is the only cyanobacterial phycobiliprotein containing phycoviolobilin as a chromophore. The phycoviolobilin chromophore is photo-reactive; upon irradiation, the chromophore undergoes a Z/E-isomerization involving the rotation of pyrrole-ring D. We have determined the structure of trimeric phycoerythrocyanin at three different experimental settings: monochromatically at 110 K and 295 K as well as with the Laue method at 288 K. Based on their chemical structures, the restraints for the phycoviolobilin of the alpha-subunit and for the phycocyanobilin chromophores of the beta-subunit were newly generated, which allows a chemically meaningful refinement of both chromophores. All three phycoerythrocyanin structures are very similar; the subunits match within 0.5 A. The detailed comparison of the data obtained with the different measurements provided information about the protein properties around the phycoviolobilin chromophore. For the first time, crystals of a phycobilisome protein are used successfully with the Laue technique. This paves the way for time-resolved macromolecular crystallography, which is able to elucidate the exact mechanisms of the phycoviolobilin photoactivity including the protein involvement.
Assuntos
Ficocianina/química , Cristalografia por Raios X , Cianobactérias/química , Isomerismo , Modelos Moleculares , Fotoquímica , Ficobilinas , Ficocianina/efeitos da radiação , Subunidades Proteicas , TermodinâmicaRESUMO
We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional Förster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Luz , Ficocianina/química , Ficocianina/efeitos da radiação , Espectrometria de Fluorescência/métodos , Relação Dose-Resposta à Radiação , Substâncias Macromoleculares/química , Substâncias Macromoleculares/efeitos da radiação , Ligação Proteica/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiaçãoRESUMO
The primary light-induced processes of phycocyanobilin were studied by means of transient-grating spectroscopy, whereby the excitation wavelength was varied over the spectral region of the ground-state absorption. On the basis of the results obtained, both the rate of the photoreaction in phycocyanobilin and the ratio of the decay of different excited-state species via two decay channels depend on the excitation wavelength. Furthermore, the formation of the photoreaction product is also dependent on the pump color. These data support a recently established model for the primary photoprocesses in phycocyanobilin. In addition, phycocyanobilin protonated at the basic pyrrolenine-type nitrogen atom was included in the transient absorption study. The decay behavior was found to be almost unchanged when compared with the unprotonated form, and this suggests that protonation of the tetrapyrrole ring structure has no effect on the overall photochemistry.
Assuntos
Proteínas de Bactérias/química , Modelos Químicos , Ficocianina/efeitos da radiação , Pirróis/efeitos da radiação , Cinética , Lasers , Fotoquímica , Ficobilinas , Ficocianina/isolamento & purificação , Conformação Proteica , Pirróis/isolamento & purificação , Spirulina , TetrapirróisRESUMO
The light state transition regulates the distribution of absorbed excitation energy between the two photosystems (PSs) of photosynthesis under varying environmental conditions and/or metabolic demands. In cyanobacteria, there is evidence for the redistribution of energy absorbed by both chlorophyll (Chl) and by phycobilin pigments, and proposed mechanisms differ in the relative involvement of the two pigment types. We assayed changes in the distribution of excitation energy with 77K fluorescence emission spectroscopy determined for excitation of Chl and phycobilin pigments, in both wild-type and state transition-impaired mutant strains of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Action spectra for the redistribution of both Chl and phycobilin pigments were very similar in both wild-type cyanobacteria. Both state transition-impaired mutants showed no redistribution of phycobilin-absorbed excitation energy, but retained changes in Chl-absorbed excitation. Action spectra for the Chl-absorbed changes in excitation in the two mutants were similar to each other and to those observed in the two wild types. Our data show that the redistribution of excitation energy absorbed by Chl is independent of the redistribution of excitation energy absorbed by phycobilin pigments and that both changes are triggered by the same environmental light conditions. We present a model for the state transition in cyanobacteria based on the x-ray structures of PSII, PSI, and allophycocyanin consistent with these results.
Assuntos
Clorofila/metabolismo , Cianobactérias/metabolismo , Ficocianina/metabolismo , Pirróis/metabolismo , Clorofila/efeitos da radiação , Cianobactérias/genética , Cianobactérias/efeitos da radiação , Luz , Modelos Biológicos , Mutação , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Ficobilinas , Ficocianina/efeitos da radiação , Pirróis/efeitos da radiação , Espectrometria de Fluorescência , TetrapirróisRESUMO
Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (lambda>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.
Assuntos
Proteínas de Bactérias/efeitos da radiação , Luz , Oxigênio/química , Proteínas de Plantas/efeitos da radiação , Compostos de Anilina , Proteínas de Bactérias/química , Deutério , Sequestradores de Radicais Livres , Concentração de Íons de Hidrogênio , Imidazóis , Indicadores e Reagentes , Complexos de Proteínas Captadores de Luz , Ficocianina/química , Ficocianina/efeitos da radiação , Ficoeritrina/química , Ficoeritrina/efeitos da radiação , Proteínas de Plantas/química , Oxigênio Singlete , Soluções , Espectrofotometria , ÁguaRESUMO
Proteins bearing colored prosthetic groups, such as the heme group in hemoglobin or the bilin group in c-phycocyanin, quench singlet oxygen by interactions at the apoprotein and the prosthetic group levels. In both proteins, chemical modification of the chromophore constitutes only a minor reaction pathway. While total deactivation of singlet oxygen takes place with rate constants of 4.0 x 10(9) and 4.2 x 10(8) M-1 s-1 for hemoglobin and phycocyanin, respectively, the bleaching of the chromophore takes place with rate constants of 3.2 x 10(6) and approximately 1 x 10(7) M-1 s-1. Irradiation of phycocyanin with red light bleaches the chromophore with low yields (approximately 0.8 x 10(-4)). Part of this bleaching is mediated by singlet oxygen produced by the irradiation of the bilin group. The low relevance of the singlet oxygen pathway is compatible with a low quantum yield (approximately 10(-3)) of free singlet oxygen production after irradiation of the protein.
Assuntos
Hemoglobinas/efeitos da radiação , Ficocianina/efeitos da radiação , Cianobactérias/química , Cianobactérias/efeitos da radiação , Hemoglobinas/química , Humanos , Técnicas In Vitro , Metemoglobina/química , Metemoglobina/efeitos da radiação , Oxigênio/química , Oxigênio/efeitos da radiação , Fotoquímica , Ficocianina/química , Oxigênio SingleteRESUMO
Visible light (>470 nm) irradiation of an oxygen-saturated solution of C-phycocyanin (C-PC) in the presence of the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. The signal intensities of DMPO-OH adduct were enhanced by superoxide dismutase (SOD) and partly inhibited by catalase. It was partly responsible for the production of DMPO-OH that superoxide anion radical (O.-2) dismutated to generate hydrogen peroxide (H2O2) which decomposed ultimately to generate the highly reactive .OH. In addition, it can be concluded that singlet oxygen (1O2) was an important intermediate according to the strong inhibitory action of 1,4-diazabicyclo[2.2.2]octane (DABCO) and histidine on DMPO-OH formation. The experimental results suggest that photodynamic action of C-PC proceed via both type I and type II mechanisms. Furthermore, the decay kinetics of DMPO-OH adduct, the effects of DMPO and C-PC concentrations as well as irradiation time on DMPO-OH adduct formation were also discussed. Concentration of C-PC should be an important factor to influence the ESR signal intensities of DMPO-OH. Therefore, it may be concluded that reasonably lower concentration of C-PC might prolong the duration of photosensitized formation of .OH and might strengthen the photodynamic action.
Assuntos
Radical Hidroxila/química , Ficocianina/química , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Luz , Micro-Ondas , Ficocianina/efeitos da radiaçãoRESUMO
The in vivo reconstitution of phycocyanobilin with apophytochrome leads to photoreversible adducts in living yeast cells. Investigations with the rice phytochrome A phycocyanobilin adduct (PHYA*) and the tobacco phytochrome B phycocyanobilin adduct (PHYB*) show that the protein stability in yeast is independent of the form of the photoreceptor. After in vivo assembly and irradiation with red light, 25.6% of the far-red light-absorbing form of PHYB* exhibited dark reversion with a half-life time of approximately 20 min. Control experiments with PHYA* revealed no dark reversion. The data indicate that the molecular basis for this reaction is the formation of heterodimers between the red and the far-red light absorbing form of phytochrome. Electron microscopic in situ localizations and in vitro sequestering experiments showed that phytochrome A was able to sequester in yeast. On the electron microscopic level, the sequestered areas of phytochrome from etiolated plants and yeast are indistinguishable. The sequestering reaction in yeast is independent of the formation of the far-red light absorbing form of phytochrome. Therefore, we discuss a new model for this reaction in plants. Since it is unlikely that yeast cells contain elements that distinguish between phytochrome A and B, we conclude that sequestering and dark reversion reflect intrinsic properties of phytochrome.
Assuntos
Ficocianina/metabolismo , Fitocromo/metabolismo , Pirróis/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Luz , Microscopia Imunoeletrônica , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiação , Ficobilinas , Ficocianina/análise , Ficocianina/efeitos da radiação , Fitocromo/genética , Fitocromo/efeitos da radiação , Plantas/genética , Plantas/metabolismo , Plantas/efeitos da radiação , Pirróis/análise , Pirróis/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , TetrapirróisRESUMO
Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.
Assuntos
Cianobactérias/química , Ficocianina/química , Ficocianina/efeitos da radiação , Fenômenos Biofísicos , Biofísica , Cianobactérias/efeitos da radiação , Transferência de Energia , Polarização de Fluorescência , Modelos Químicos , Fotoquímica , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
Raman spectra of phycocyanobilin, phycocyanin, and allophycocyanin were obtained at resonance with their visible and near-UV transitions. These spectra were empirically assigned with the help of 14N- and 15N-isotopic substitutions and comparisons with resonance Raman spectra of phycoerythrin. These results confirm the previously suggested assignment of a conformation-sensitive band around 1239-1246 cm-1 to a mode involving nu CmH and nu CN coordinates. Computer-assisted decomposition of the complex, conformation-sensitive 1580-1670-cm-1 region yielded five components that we labeled I-V. The previously described spectral changes observed upon monomerization and denaturation in resonance Raman spectra of phycocyanin and allophycocyanin essentially arise from changes in the relative intensities of these components. Component I (around 1649-1651 cm-1) and component III (1621-1624 cm-1) originate predominantly from nu C=C at C15 of the chromophore. Their relative intensity ratio reflects the relative amounts of C15-Z-anti and C15-Z-syn methine bridge conformations, respectively. Component II (1633-1638 cm-1) is ascribed to a nu C=C mode of pyrrole rings; it is not sensitive to the chromophore conformation. Component IV is also conformation-insensitive and originates from nu C=N and nu C=C coordinates, most likely from ring C. Component V (1591-1594 cm-1) involves a nu C=N coordinate in ring D, coupled to a nu C=C coordinate of the C15 methine bridge. The implications of the present assignments on those of resonance Raman active modes of phytochrome are discussed. A consistent set of correlations between chromophore conformations and resonance Raman data is obtained for both phycobiliproteins and phytochrome.
Assuntos
Ficocianina/química , Pirróis/química , Cianobactérias/química , Luz , Computação Matemática , Conformação Molecular , Isótopos de Nitrogênio , Ficobilinas , Ficocianina/efeitos da radiação , Ficoeritrina/química , Ficoeritrina/efeitos da radiação , Desnaturação Proteica , Pirróis/efeitos da radiação , Análise Espectral Raman , Tetrapirróis , Raios UltravioletaRESUMO
The UV survival characteristics of a wild-type and a UV resistant strain of Gloeocapsa alpicola were compared. Except for a higher carotenoid content the resistant strain was similar to the wild-type. However, on exposure to UV radiation the level of carotenoids in the wild-type fell sharply whereas no such decrease occurred in the resistant strain. Growth of cells in diphenylamine and under red and blue light reduced the survival rate. This phenomenon is linked with a reduced carotenoid level in the cell, indicating that they perform an important protective function.