Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
FEMS Microbiol Ecol ; 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31778156

RESUMO

The diversity of cyanobacteria along the Alaskan North Slope was investigated. We isolated and cultivated 57 strains of cyanobacteria and sequenced a section of their rRNA operon containing a fragment of the 16S rRNA gene. Here, we describe 17 found species belonging mainly to families Coleofasciculaceae, Microcoleaceae, Oculatellaceae, Leptolyngbyaceae and to the order Synechococcales. In pursuing a conservative polyphasic approach, we utilized suggested thresholds in 16S rRNA gene differences in parallel with morphological differences between new and already described taxa for description of new species and genera. Based on the combination of morphological, molecular and ecological analysis on collected and cultured strains we describe two genera Gibliniella and Shackletoniella as well as six cyanobacterial species; Cephalothrix alaskaensis, Tildeniella alaskaensis, Pseudophormidium americanum, Leptodesmis alaskaensis, Albertania alaskaensis and Nodosilinea alaskaensis. Here, a polyphasic approach was used to identify 8 novel and 9 established cyanobacterial taxa from a previously non-investigated region that uncovered a high degree of biodiversity in extreme polar environments.

2.
Photosynth Res ; 142(2): 181-193, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31267356

RESUMO

The influence of temperature on photosynthetic reactions was investigated by a combination of time-resolved bacteriochlorophyll fluorescence, steady-state and differential absorption spectroscopy, and polarographic respiration measurements in intact cells of purple non-sulphur bacterium Rhodospirillum rubrum. Using variable bacteriochlorophyll fluorescence, it was found that the electron-transport activity increased with the increasing temperature up to 41 °C. The fast and medium components of the fluorescence decay kinetics followed the ideal Arrhenius equation. The calculated activation energy for the fast component was Ea1 = 16 kJ mol-1, while that of the medium component was more than double, with Ea2 = 38 kJ mol-1. At temperatures between 41 and 59 °C, the electron transport was gradually, irreversibly inhibited. Interestingly, the primary charge separation remained fully competent from 20 to 59 °C as documented by both BChl fluorescence and differential absorption spectroscopy of the P870+ signal. At temperatures above 60 °C, the primary photochemistry became reversibly inhibited, which was manifested by an increase in minimal fluorescence, F0, whereas maximal fluorescence, FM, slowly declined. Finally, above 71 °C, the photosynthetic complexes began to disassemble as seen in the decline of all fluorometric parameters and the disappearance of the LH1 absorption band at 880 nm. The extended optimal temperature of photosynthetic reaction centre in a model species of Rhodospirillales adds on the evidence that the good thermostability of the photosynthetic reaction centres is present across all Alphaproteobacteria.

3.
Biochim Biophys Acta Bioenerg ; 1860(8): 640-650, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226316

RESUMO

Magnesium (Mg2+) is the ubiquitous metal ion present in chlorophyll and bacteriochlorophyll (BChl), involved in photosystems in photosynthetic organisms. In the present study we investigated targets of toxic copper binding to the photosynthetic apparatus of the anoxygenic purple bacterium Rhodospirillum rubrum. This was done by a combination of in vivo measurements of flash photolysis and fast fluorescence kinetics combined with the analysis of metal binding to pigments and pigment-protein complexes isolated from Cu-stressed cells by HPLC-ICPMS (ICP-sfMS). This work concludes that R. rubrum is highly sensitive to Cu2+, with a strong inhibition of the photosynthetic reaction centres (RCs) already at 2 µM Cu2+. The inhibition of growth and of RC activity was related to the formation of Cu-containing BChl degradation products that occurred much more in the RC than in LH1. These results suggest that the shift of metal centres in BChl from Mg2+ to Cu2+ can occur in vivo in the RCs of R. rubrum under environmentally realistic Cu2+ concentrations, leading to a strong inhibition of the function of these RCs.


Assuntos
Cobre/toxicidade , Complexo de Proteínas do Centro de Reação Fotossintética/antagonistas & inibidores , Rhodospirillum rubrum/efeitos dos fármacos , Bacterioclorofilas/química , Complexos de Proteínas Captadores de Luz , Magnésio
4.
Sci Rep ; 9(1): 7367, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089169

RESUMO

Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome-wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.

5.
Spectrochim Acta A Mol Biomol Spectrosc ; 212: 262-271, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30658280

RESUMO

We tested the potential of Raman microspectroscopy to determine carotenoid pigments - both primary (lutein, beta-carotene) and secondary (astaxanthin) carotenoids - in the different species and life-cycle stages of snow algae from the order Chlamydomonadales (Chlorophyta). We compared the performance of Raman spectrometry to a reference method of biological pigment analysis, high-performance liquid chromatography (HPLC). The three main carotenoid Raman bands of the astaxanthin-rich red cysts were located at 1520, 1156 and 1006 cm-1. The shifts (orange aplanozygotes and green motile cells with flagella) in the position of the ν1(CC) Raman band of the polyenic chain is consistent with the expected changes in the ratios of the various carotenoid pigments. Flagellated green cells commonly contain lutein as a major carotenoid, together with minor amounts of ß­carotene and varying amounts of antheraxanthin, violaxanthin and neoxanthin. Aplanozygotes contain mixtures of both primary and secondary carotenoids. In most cases, the ν1(CC) band is an overlapping set of bands, which is due to the signal of all carotenoid pigments in the sample, and a deconvolution along with the band position shifts (mainly ν1) could be used to characterize the mixture of carotenoids. However, the ability of Raman spectroscopy to discriminate between structurally slightly differing carotenoid pigments or several carotenoids in an admixture in an unknown biological system remains limited.


Assuntos
Carotenoides/análise , Cromatografia Líquida de Alta Pressão/métodos , Neve , Análise Espectral Raman/métodos , Clorofila/análise , Clorofila A/análise , Europa (Continente) , Geografia , Microalgas , Microespectrofotometria
6.
Extremophiles ; 23(1): 35-48, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30284641

RESUMO

Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S rRNA gene amplicon sequencing. Altogether, 34 sites were investigated along the 71-39 °C temperature gradient. Analysis of samples from eight representative sites shown that Illumina, optical microscopy, and Roche 454 identified 72, 45 and 19% respective occurrences of all cumulatively present taxa. Optical microscopy failed to detect species of minor occurrence; whereas, amplicon sequencing technologies suffered from failed primer annealing and the presence of species with extensive extracellular polysaccharides production. Amplicon sequencing of the 16S rRNA gene V5-V6 region performed by Illumina identified the cyanobacteria most reliably to the generic level. Nevertheless, only the combined use of optical microscopy, cultivation and sequencing methods allowed for reliable estimate of the cyanobacterial diversity. Here, we show that Rupite hot-spring system hosts one of the richest cyanobacterial flora reported from a single site above 50 °C. Chlorogloeopsis sp. was the most abundant at the highest temperature (68 °C), followed by Leptolyngbya boryana, Thermoleptolyngbya albertanoae, Synechococcus bigranulatus, Oculatella sp., and Desertifilum sp. thriving above 60 °C, while Leptolyngbya geysericola, Geitlerinema splendidum, and Cyanobacterium aponinum were found above 50 °C.


Assuntos
Cianobactérias/genética , Fontes Termais/microbiologia , Microbiota , Cianobactérias/classificação , Cianobactérias/citologia , Cianobactérias/isolamento & purificação , RNA Ribossômico 16S/genética
7.
Environ Microbiol ; 20(2): 724-733, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29159858

RESUMO

Aerobic anoxygenic photosynthetic bacteria are an important component of marine microbial communities. They produce energy in light using bacteriochlorophyll a containing photosystems. This extra energy provides an advantage over purely heterotrophic bacteria. One of the most intensively studied AAP bacteria is Dinoroseobacter shibae, a member of the environmentally important Roseobacter clade. Light stimulates its growth and metabolism, but the effect of light intensity remains unclear. Here, we show that an increase in biomass along an irradiance gradient followed the exponential rise to the maximum curve, with saturation at about 300 µmol photons m-2 s-1 , without any inhibition at light intensities up to 600 µmol photons m-2 s-1 . The cells adapted to higher irradiance by reducing pigmentation and increasing the electron transfer rate. This additional energy allowed D. shibae to redirect the metabolism of organic carbon sources such as glucose, leucine, glutamate, acetate and pyruvate toward anabolism, resulting in a twofold increase of their assimilation rates. We provide equations that can be feasibly incorporated into the existing model of D. shibae metabolism to further advance our understanding of the role of photoheterotrophy in the ocean.


Assuntos
Bacterioclorofila A/metabolismo , Transporte de Elétrons/fisiologia , Metabolismo Energético/fisiologia , Compostos Orgânicos/metabolismo , Fotossíntese/fisiologia , Roseobacter/metabolismo , Organismos Aquáticos/metabolismo , Biomassa , Luz
8.
PLoS Biol ; 15(12): e2003943, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29253871

RESUMO

The majority of life on Earth depends directly or indirectly on the sun as a source of energy. The initial step of photosynthesis is facilitated by light-harvesting complexes, which capture and transfer light energy into the reaction centers (RCs). Here, we analyzed the organization of photosynthetic (PS) complexes in the bacterium G. phototrophica, which so far is the only phototrophic representative of the bacterial phylum Gemmatimonadetes. The isolated complex has a molecular weight of about 800 ± 100 kDa, which is approximately 2 times larger than the core complex of Rhodospirillum rubrum. The complex contains 62.4 ± 4.7 bacteriochlorophyll (BChl) a molecules absorbing in 2 distinct infrared absorption bands with maxima at 816 and 868 nm. Using femtosecond transient absorption spectroscopy, we determined the energy transfer time between these spectral bands as 2 ps. Single particle analyses of the purified complexes showed that they were circular structures with an outer diameter of approximately 18 nm and a thickness of 7 nm. Based on the obtained, we propose that the light-harvesting complexes in G. phototrophica form 2 concentric rings surrounding the type 2 RC. The inner ring (corresponding to the B868 absorption band) is composed of 15 subunits and is analogous to the inner light-harvesting complex 1 (LH1) in purple bacteria. The outer ring is composed of 15 more distant BChl dimers with no or slow energy transfer between them, resulting in the B816 absorption band. This completely unique and elegant organization offers good structural stability, as well as high efficiency of light harvesting. Our results reveal that while the PS apparatus of Gemmatimonadetes was acquired via horizontal gene transfer from purple bacteria, it later evolved along its own pathway, devising a new arrangement of its light harvesting complexes.


Assuntos
Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Fotossíntese/fisiologia , Bactérias/classificação , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Transferência Genética Horizontal , Filogenia
9.
Sci Rep ; 7: 44580, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300167

RESUMO

Interquinone QA- → QB electron-transfer (ET) in isolated photosystem II reaction centers (PSII-RC) is protein-gated. The temperature-dependent gating frequency "k" is described by the Eyring equation till levelling off at T ≥ 240 °K. Although central to photosynthesis, the gating mechanism has not been resolved and due to experimental limitations, could not be explored in vivo. Here we mimic the temperature dependency of "k" by enlarging VD1-208, the volume of a single residue at the crossing point of the D1 and D2 PSII-RC subunits in Synechocystis 6803 whole cells. By controlling the interactions of the D1/D2 subunits, VD1-208 (or 1/T) determines the frequency of attaining an ET-active conformation. Decelerated ET, impaired photosynthesis, D1 repair rate and overall cell physiology upon increasing VD1-208 to above 130 Å3, rationalize the >99% conservation of small residues at D1-208 and its homologous motif in non-oxygenic bacteria. The experimental means and resolved mechanism are relevant for numerous transmembrane protein-gated reactions.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/química , Synechocystis/química , Respiração Celular/genética , Transporte de Elétrons/genética , Elétrons , Cinética , Luz , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema II/genética , Synechocystis/genética
11.
Int J Syst Evol Microbiol ; 65(8): 2410-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25899503

RESUMO

A red-pigmented, bacteriochlorophyll (BChl) a-producing strain, AP64T, was isolated previously from the freshwater Swan Lake located in the western Gobi Desert. Based on its 16S rRNA gene sequence identity (96.1%) to the type strain Gemmatimonas aurantiaca T-27T, the new isolate was tentatively classified as a member of the bacterial phylum Gemmatimonadetes. Here, we report its formal description and polyphasic characterization. Strain AP64T grew best on agar media under 9.8-15.2% atmospheric oxygen. The cells were rods, dividing by symmetrical or asymmetrical binary fission. Budding structures were also observed. Its genomic DNA G+C content was 64.4% (from the draft genome sequence). Phylogenetic analysis based on the 16S rRNA gene sequence clearly separated AP64T from related species. Its genotypic differentiation from phylogenetically close relatives was further supported by performing in silico DNA-DNA hybridization and calculating average nucleotide identity, whereas the high percentage (67.3%) of shared conserved proteins between strain AP64T and Gemmatimonas aurantiaca T-27T supports the classification of the two strains into the same genus. Strain AP64T contained C16 : 1, C14 : 1 and C18 : 1ω9c as predominant fatty acids. The main respiratory quinone was menaquinone 8 (MK-8). The most distinctive feature of strain AP64T was the presence of fully functional purple bacterial photosynthetic reaction centres. The main CO2-fixation pathways were absent. Strain AP64T was capable of growth and BChl production in constant darkness. Thus, strain AP64T is a facultatively photoheterotrophic organism. It represents a novel species of the genus Gemmatimonas, for which the name Gemmatimonasphototrophica sp. nov. is proposed. The type strain is AP64T ( = DSM 29774T = MCCC 1K00454T). Emended descriptions of the genus Gemmatimonas and Gemmatimonas aurantiaca are also provided.


Assuntos
Bactérias/classificação , Lagos/microbiologia , Filogenia , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana , Bacterioclorofila A/química , Composição de Bases , Sequência de Bases , China , DNA Bacteriano/genética , Clima Desértico , Ácidos Graxos/química , Água Doce/microbiologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
12.
Am J Respir Crit Care Med ; 190(5): 522-32, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25029038

RESUMO

RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.


Assuntos
Agonistas do Canal de Sódio Epitelial/metabolismo , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/metabolismo , Alvéolos Pulmonares/metabolismo , Edema Pulmonar/metabolismo , Estreptolisinas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Bactérias , Agonistas do Canal de Sódio Epitelial/química , Canais Epiteliais de Sódio/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peptídeos Cíclicos/química , Alvéolos Pulmonares/microbiologia , Edema Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/química
13.
FEMS Microbiol Ecol ; 89(2): 303-15, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24698015

RESUMO

Here, we report an effect of short acclimation to a wide span of temperatures on photosynthetic electron transfer, lipid and fatty acid composition in the snow alga Chlamydomonas cf. nivalis. The growth and oxygen evolution capacity were low at 2 °C yet progressively enhanced at 10 °C and were significantly higher at temperatures from 5 to 15 °C in comparison with the mesophilic control Chlamydomonas reinhardtii. In search of the molecular mechanisms responsible for the adaptation of photosynthesis to low temperatures, we have found unprecedented high rates of QA to QB electron transfer. The thermodynamics of the process revealed the existence of an increased structural flexibility that we explain with the amino acid changes in the D1 protein combined with the physico-chemical characteristics of the thylakoid membrane composed of > 80% negatively charged phosphatidylglycerol.


Assuntos
Chlamydomonas/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Adaptação Fisiológica , Sequência de Aminoácidos , Chlamydomonas/crescimento & desenvolvimento , Temperatura Baixa , Sequência Conservada , Transporte de Elétrons , Membranas Intracelulares/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Dados de Sequência Molecular , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Quinonas/metabolismo , Temperatura Ambiente , Termodinâmica , Tilacoides/metabolismo
14.
J Phys Chem B ; 117(38): 10987-99, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-23130956

RESUMO

Light-harvesting complexes ensure necessary flow of excitation energy into photosynthetic reaction centers. In the present work, transient absorption measurements were performed on LH1-RC complexes isolated from two aerobic anoxygenic phototrophs (AAPs), Roseobacter sp. COL2P containing the carotenoid spheroidenone, and Erythrobacter sp. NAP1 which contains the carotenoids zeaxanthin and bacteriorubixanthinal. We show that the spectroscopic data from the LH1-RC complex of Roseobacter sp. COL2P are very similar to those previously reported for Rhodobacter sphaeroides, including the transient absorption spectrum originating from the intramolecular charge-transfer (ICT) state of spheroidenone. Although the ICT state is also populated in LH1-RC complexes of Erythrobacter sp. NAP1, its appearance is probably related to the polarity of the bacteriorubixanthinal environment rather than to the specific configuration of the carotenoid, which we hypothesize is responsible for populating the ICT state of spheroidenone in LH1-RC of Roseobacter sp. COL2P. The population of the ICT state enables efficient S1/ICT-to-bacteriochlorophyll (BChl) energy transfer which would otherwise be largely inhibited for spheroidenone and bacteriorubixanthinal due to their low energy S1 states. In addition, the triplet states of these carotenoids appear well-tuned for efficient quenching of singlet oxygen or BChl-a triplets, which is of vital importance for oxygen-dependent organisms such as AAPs.


Assuntos
Proteínas de Bactérias/química , Carotenoides/química , Complexos de Proteínas Captadores de Luz/química , Roseobacter/metabolismo , Xantofilas/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Transferência de Energia , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Rhodobacter sphaeroides/metabolismo , Sphingomonadaceae/metabolismo , Zeaxantinas
15.
PLoS One ; 7(10): e46694, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23071614

RESUMO

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.


Assuntos
Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Spinacia oleracea/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Ligações de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise Espectral Raman
16.
Curr Pharm Des ; 18(27): 4236-43, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22697478

RESUMO

The lectin-like domain of Tumor Necrosis Factor (TNF), mimicked by the TIP peptide, activates amiloride-sensitive sodium uptake in type II alveolar epithelial cells and as such increases alveolar liquid clearance in dysfunctional lungs. This protective effect is blunted upon mutation of residues T105, E107 and E110 in human TNF into alanine or upon pre-incubation of the cytokine with the disaccharide N,N'-diacetylchitobiose. In this study, we used molecular docking and molecular dynamics simulation to predict the binding sites for N,N'-diacetylchitobiose and trimannose-O-ethyl in the lectin-like domain of TNF and in the TIP peptide. Specific sites (K98, S99, P100, Q102 and E116) in the three loops of the lectin-like domain provide specific binding for both oligosaccharides, but none of the residues crucial for anti-edema activity are involved in hydrogen bonding with oligosaccharides or are subjected to steric hindrance by them. These results thus suggest that neither chitobiose nor trimannose affect crucial amino acids, while they occupy the cavity in the lectin-like domain. Consequently, both crucial amino acids and the emptiness of the cavity in the lectin-like domain may be critical for TNF's lectin-like activity. Analogously, the R4, E5, P7, Y16 amino acids of the TIP peptide are involved in forming hydrogen bonds with both oligosaccharides, whereas residues T6, E8 and E11 (corresponding to T105, E107 and E110 in hTNF) play an important role in stabilizing the peptide-oligosaccharide complex, supporting the hypothesis that amino acids in the polar region (TPEGAE) of the TIP peptide represent only a partial binding motif for sugars.


Assuntos
Dissacarídeos/metabolismo , Oligossacarídeos/metabolismo , Peptídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Humanos , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Peptídeos/química , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética
17.
PLoS One ; 6(12): e28389, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22216094

RESUMO

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43 °C but only at elevated CO(2) (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated Q(A)(-)→Q(B) electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO(2) concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.


Assuntos
Adaptação Fisiológica , Dióxido de Carbono/análise , Cianobactérias/fisiologia , Temperatura Alta , Mutação , Complexo de Proteína do Fotossistema II/genética , Cianobactérias/genética , Genes Bacterianos , Luz
18.
Ultramicroscopy ; 109(8): 1056-60, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19362420

RESUMO

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.


Assuntos
Células Eucarióticas/ultraestrutura , Microscopia de Força Atômica/métodos , Animais , Elasticidade , Células Endoteliais/ultraestrutura , Células Epiteliais/ultraestrutura , Humanos , Pulmão/citologia , Camundongos , Miocárdio/citologia
19.
Nature ; 442(7104): 827-30, 2006 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-16862124

RESUMO

Adjustment of catalytic activity in response to diverse ambient temperatures is fundamental to life on Earth. A crucial example of this is photosynthesis, where solar energy is converted into electrochemical potential that drives oxygen and biomass generation at temperatures ranging from those of frigid Antarctica to those of scalding hot springs. The energy conversion proceeds by concerted mobilization of electrons and protons on photoexcitation of reaction centre protein complexes. Following physicochemical paradigms, the rates of imperative steps in this process were predicted to increase exponentially with rising temperatures, resulting in different yields of solar energy conversion at the distinct growth temperatures of photosynthetic mesophiles and extremophiles. In contrast, here we show a meticulous adjustment of energy conversion rate, resulting in similar yields from mesophiles and thermophiles. The key molecular players in the temperature adjustment process consist of a cluster of hitherto unrecognized protein cavities and an adjacent packing motif that jointly impart local flexibility crucial to the reaction centre proteins. Mutations within the packing motif of mesophiles that increase the bulkiness of the amino-acid side chains, and thus reduce the size of the cavities, promote thermophilic behaviour. This novel biomechanical mechanism accounts for the slowing of the catalytic reaction above physiological temperatures in contradiction to the classical Arrhenius paradigm. The mechanism provides new guidelines for manipulating the acclimatization of enzymes to the ambient temperatures of diverse habitats. More generally, it reveals novel protein elements that are of potential significance for modulating structure-activity relationships in membrane and globular proteins alike.


Assuntos
Aclimatação , Fotossíntese , Proteínas/química , Proteínas/metabolismo , Temperatura Ambiente , Clorofila/metabolismo , Sequência Conservada , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Elétrons , Transferência de Energia , Modelos Moleculares , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Maleabilidade , Conformação Proteica , Proteobactérias/química , Proteobactérias/metabolismo , Proteobactérias/efeitos da radiação , Relação Estrutura-Atividade
20.
Micron ; 36(6): 483-502, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15951188

RESUMO

Progress in various fields of microscopy techniques brought up enormous possibilities to study the photosynthesis down to the level of individual pigment-protein complexes. The aim of this review is to present recent developments in the photosynthesis research obtained using such highly advanced techniques. Three areas of microscopy techniques covering optical microscopy, electron microscopy and scanning probe microscopy are reviewed. Whereas the electron microscopy and scanning probe microscopy are used in photosynthesis mainly for structural studies of photosynthetic pigment-protein complexes, the optical microscopy is used also for functional studies.


Assuntos
Microscopia , Fotossíntese , Cristalografia , Complexos de Proteínas Captadores de Luz/metabolismo , Microscopia Eletrônica , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Análise Espectral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA