RESUMO
In photosynthetic thylakoid membranes the proton motive force (pmf) not only drives ATP synthesis, in addition it is central to controlling and regulating energy conversion. As a consequence, dynamic fine-tuning of the two pmf components, electrical (Δψ) and chemical (ΔpH), is an essential element for adjusting photosynthetic light reactions to changing environmental conditions. Good evidence exists that the Δψ/ΔpH partitioning is controlled by thylakoid potassium and chloride ion transporters and channels. However, a detailed mechanistic understanding of how these thylakoid ion transporter/channels control pmf partitioning is lacking. Here, we combined functional measurements on potassium and chloride ion transporter and channel loss-of-function mutants with extended mathematical simulations of photosynthetic light reactions in thylakoid membranes to obtain detailed kinetic insights into the complex interrelationship between membrane energization and ion fluxes across thylakoid membranes. The data reveal that potassium and chloride fluxes in the thylakoid lumen determined by the K+/H+ antiporter KEA3 and the voltage-gated Cl- channel VCCN1/Best1 have distinct kinetic responses that lead to characteristic and light-intensity-dependent Δψ/ΔpH oscillations. These oscillations fine-tune photoprotective mechanisms and electron transport which are particularly important during the first minutes of illumination and under fluctuating light conditions. By employing the predictive power of the model, we unravelled the functional consequences of changes in KEA3 and VCCN1 abundance and regulatory/enzymatic parameters on membrane energization and photoprotection.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Transporte de Elétrons/fisiologia , Concentração de Íons de Hidrogênio , Fotossíntese/fisiologia , Força Próton-Motriz/fisiologia , Tilacoides/fisiologia , Transporte de Elétrons/genética , Variação Genética , Genótipo , Mutação , Fotossíntese/genética , Força Próton-Motriz/genética , Tilacoides/genéticaRESUMO
In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.
Assuntos
Antiporters/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Subunidades Proteicas/metabolismo , Força Próton-Motriz/fisiologia , Sequência de Aminoácidos , Antiporters/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Prótons , Sais/química , Thermus thermophilus/metabolismo , Água/químicaRESUMO
Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host's existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA.IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Força Próton-Motriz/genética , Força Próton-Motriz/fisiologiaRESUMO
In forests, understory plants are usually exposed to sunflecks on timescales of seconds or minutes. However, it is unclear how understory plants acclimate to fluctuating light. In this study, we compared chlorophyll fluorescence, PSI redox state and the electrochromic shift signal under fluctuating light between an understory plant Paris polyphylla (Liliaceae) and a light-demanding plant Bletilla striata (Orchidaceae). Within the first seconds after transition from low to high light, PSI was highly oxidized in P. polyphylla but was highly reduced in B. striata, although both species could not generate a sufficient trans-thylakoid proton gradient (ΔpH). Furthermore, the outflow of electrons from PSI to O2 was not significant in P. polyphylla, as indicated by the P700 redox kinetics upon dark-to-light transition. Therefore, the different responses of PSI to fluctuating light between P. polyphylla and B. striata could not be explained by ΔpH formation or alternative electron transport. In contrast, upon a sudden transition from low to high light, electron flow from PSII was much lower in P. polyphylla than in B. striata, suggesting that the rapid oxidation of PSI in P. polyphylla was largely attributed to the lower PSII activity. We propose, for the first time, that down-regulation of PSII activity is an important strategy used by some understory angiosperms to cope with sunflecks.
Assuntos
Luz , Melanthiaceae/enzimologia , Complexo de Proteína do Fotossistema II/metabolismo , Força Próton-Motriz/fisiologia , Tilacoides/enzimologia , Transporte de Elétrons/fisiologia , Orchidaceae/enzimologia , Oxigênio/metabolismoRESUMO
The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2 formation and the generation of a transmembrane electrochemical ion gradient ([Formula: see text]). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across the membrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.
Assuntos
Fenômenos Bioquímicos , Redes e Vias Metabólicas , Oxirredutases/metabolismo , Força Próton-Motriz/fisiologia , Thermoanaerobacter/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeído Oxirredutases/metabolismo , Ciclo do Carbono , Monóxido de Carbono/metabolismo , Membrana Celular/metabolismo , Hidrogênio/metabolismo , Complexos Multienzimáticos/metabolismo , Família Multigênica , Oxirredução , Vesículas Secretórias/metabolismo , Sódio/metabolismo , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genéticaRESUMO
Bacterial type 4 pili (T4P) belong to the strongest molecular machines. The gonococcal T4P retraction ATPase PilT supports forces exceeding 100 pN during T4P retraction. Here, we address the question of whether gonococcal T4P retract in the absence of PilT. We show that pilT deletion strains indeed retract their T4P, but the maximum force is reduced to 5 pN. Similarly, the speed of T4P retraction is lower by orders of magnitude compared to that of T4P retraction driven by PilT. Deleting the pilT paralogue pilT2 further reduces the speed of T4P retraction, yet T4P retraction is detectable in the absence of all three pilT paralogues. Furthermore, we show that depletion of proton motive force (PMF) slows but does not inhibit pilT-independent T4P retraction. We conclude that the retraction ATPase is not essential for gonococcal T4P retraction. However, the force generated in the absence of PilT is too low to support important functions of T4P, including twitching motility, fluidization of colonies, and induction of host cell response.IMPORTANCE Bacterial type 4 pili (T4P) have been termed the "Swiss Army knives" of bacteria because they perform numerous functions, including host cell interaction, twitching motility, colony formation, DNA uptake, protein secretion, and surface sensing. The pilus fiber continuously elongates or retracts, and these dynamics are functionally important. Curiously, only a subset of T4P systems employ T4P retraction ATPases to power T4P retraction. Here, we show that one of the strongest T4P machines, the gonococcal T4P, retracts without a retraction ATPase. Biophysical characterization reveals strongly reduced force and speed compared to retraction with ATPase. We propose that bacteria encode retraction ATPases when T4P have to generate high-force-supporting functions like twitching motility, triggering host cell response, or fluidizing colonies.
Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Proteínas Motores Moleculares/metabolismo , Neisseria gonorrhoeae/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Fímbrias/metabolismo , Força Próton-Motriz/fisiologiaRESUMO
The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome bc1-cytochrome c oxidase supercomplex and have been proposed to play a role in the assembly and/or modulation of the activity of the cytochrome c oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In this study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV and limits the level of CIV activity required for maintenance of the PMF and growth under aerobic conditions.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Consumo de Oxigênio/fisiologia , Força Próton-Motriz/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.
Assuntos
Mitocôndrias/química , Bombas de Próton/fisiologia , Análise Espectral/métodos , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Camundongos , Mitocôndrias/metabolismo , Oxirredução , Consumo de Oxigênio , Força Próton-Motriz/fisiologia , Células RAW 264.7RESUMO
This study described a pH-gradient dissolution method combined with flux measurements as an in vitro tool for assessing the risk of bioavailability reduction due to drug-drug interactions (DDI) caused by acid reducing agents (ARAs). The device incorporates absorption chambers into USP II dissolution vessels, with fiber optic UV-probes monitoring concentration in situ. Dosage forms of Genentech BCS class II drugs, GDC-0810, GDC-0941, and compound A, were tested by starting the dissolution in either pH 1.6 or pH 4.0 media then converting to FaSSIF after 30 min. GDC-0810 showed no significant difference in flux between the two conversion experiments. A supersaturation phase was observed for GDC-0941 in the pH 1.6 experiments after media conversion to FaSSIF; however, it did not appear to occur in the pH 4.0 experiment due to low drug solubility at pH 4.0, resulting in a 95% decrease in flux compared to pH 1.6 experiment. The extent of flux reduction and the total accumulated API mass in the absorption chamber agreed well with the 89% reduction in mean Cmax and the 82% reduction in mean AUC from dog PK study between animals treated with pentagastrin and famotidine. Testing of the compound A optimized formulation tablets showed a 25% reduction in flux and in vitro absorbed amount by changing pH 1.6 to 4.0, correlating well with the AUC decrease in clinical studies. Good correlation between in vitro data and in vivo PK data demonstrated the applicability of the method for formulators to develop drug products mitigating DDI from ARAs.
Assuntos
Cinamatos/química , Cinamatos/farmacocinética , Indazóis/química , Indazóis/farmacocinética , Sulfonamidas/química , Sulfonamidas/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Cães , Interações Medicamentosas/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Força Próton-Motriz/efeitos dos fármacos , Força Próton-Motriz/fisiologia , Solubilidade , ComprimidosRESUMO
In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.
Assuntos
Camellia/fisiologia , Folhas de Planta , Força Próton-Motriz/fisiologia , Tilacoides , Camellia/classificação , Clorofila/fisiologia , Fluorescência , Fenótipo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Folhas de Planta/fisiologia , Transpiração Vegetal , ATPases Translocadoras de Prótons/fisiologia , Tilacoides/fisiologiaRESUMO
BACKGROUND: pH is frequently reported as the main driver for prokaryotic community structure in soils. However, pH changes are also linked to "spillover effects" on other chemical parameters (e.g., availability of Al, Fe, Mn, Zn, and Cu) and plant growth, but these indirect effects on the microbial communities are rarely investigated. Usually, pH also co-varies with some confounding factors, such as land use, soil management (e.g., tillage and chemical inputs), plant cover, and/or edapho-climatic conditions. So, a more comprehensive analysis of the direct and indirect effects of pH brings a better understanding of the mechanisms driving prokaryotic (archaeal and bacterial) community structures. RESULTS: We evaluated an agricultural soil pH gradient (from 4 to 6, the typical range for tropical farms), in a liming gradient with confounding factors minimized, investigating relationships between prokaryotic communities (16S rRNA) and physical-chemical parameters (indirect effects). Correlations, hierarchical modeling of species communities (HMSC), and random forest (RF) modeling indicated that both direct and indirect effects of the pH gradient affected the prokaryotic communities. Some OTUs were more affected by the pH changes (e.g., some Actinobacteria), while others were more affected by the indirect pH effects (e.g., some Proteobacteria). HMSC detected a phylogenetic signal related to the effects. Both HMSC and RF indicated that the main indirect effect was the pH changes on the availability of some elements (e.g., Al, Fe, and Cu), and secondarily, effects on plant growth and nutrient cycling also affected the OTUs. Additionally, we found that some of the OTUs that responded to pH also correlated with CO2, CH4, and N2O greenhouse gas fluxes. CONCLUSIONS: Our results indicate that there are two distinct pH-related mechanisms driving prokaryotic community structures, the direct effect and "spillover effects" of pH (indirect effects). Moreover, the indirect effects are highly relevant for some OTUs and consequently for the community structure; therefore, it is a mechanism that should be further investigated in microbial ecology.
Assuntos
Archaea/classificação , Bactérias/classificação , Fenômenos Ecológicos e Ambientais/fisiologia , Força Próton-Motriz/fisiologia , Solo/química , Archaea/genética , Bactérias/genética , Sequência de Bases , Brasil , Gases de Efeito Estufa/análise , Sequenciamento de Nucleotídeos em Larga Escala , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S/genética , Microbiologia do SoloRESUMO
This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG's conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Trifosfato de Adenosina/metabolismo , Eletrofisiologia , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Methanocaldococcus/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Força Próton-Motriz/fisiologiaRESUMO
The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.
Assuntos
Campylobacter jejuni/metabolismo , Força Próton-Motriz/fisiologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Formiatos/metabolismo , Hidrogênio , Potenciais da Membrana , Oxirredução , Oxigênio , FosforilaçãoRESUMO
The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Força Próton-Motriz/fisiologia , ATPases Translocadoras de Prótons/metabolismoRESUMO
Plants use sunlight as their primary energy source. During photosynthesis, absorbed light energy generates reducing power by driving electron transfer reactions. These are coupled to the transfer of protons into the thylakoid lumen, generating a proton motive force (pmf) required for ATP synthesis. Sudden alterations in light availability have to be met by regulatory mechanisms to avoid the over-accumulation of reactive intermediates and maximize energy efficiency. Here, the acidification of the lumen, as an intermediate product of photosynthesis, plays an important role by regulating photosynthesis in response to excitation energy levels. Recent findings reveal pmf regulation and the modulation of its composition as key determinants for efficient photosynthesis, plant growth, and survival in fluctuating light environments.
Assuntos
Cloroplastos/metabolismo , Luz , Força Próton-Motriz/fisiologia , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Cloroplastos/efeitos da radiação , Transporte de Elétrons/fisiologia , Transporte de Elétrons/efeitos da radiação , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiaçãoRESUMO
A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high-throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high-throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.
Assuntos
Cromatografia por Troca Iônica , Proteínas/química , Força Próton-Motriz/fisiologia , Cloreto de Sódio/química , Anticorpos Monoclonais/química , Soluções Tampão , Resinas de Troca de Cátion , Concentração de Íons de Hidrogênio , Modelos Químicos , Ligação Proteica , Proteínas/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Força Próton-Motriz/fisiologia , Simportadores/metabolismo , Transporte Biológico Ativo/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas de Transporte de Monossacarídeos/genética , Simportadores/genéticaRESUMO
In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl(-)) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl(-) channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Canais de Cloreto/metabolismo , Proteínas Mitocondriais/metabolismo , Fotossíntese/fisiologia , Fatores de Processamento de RNA/metabolismo , Proteínas de Arabidopsis/genética , Cloretos/metabolismo , Transporte de Elétrons/fisiologia , Fenômenos Eletrofisiológicos , Luz , Microscopia Eletrônica de Transmissão , Proteínas Mitocondriais/genética , Mutação , Plantas Geneticamente Modificadas/fisiologia , Força Próton-Motriz/fisiologia , Fatores de Processamento de RNA/genética , Tilacoides/metabolismo , Tilacoides/ultraestruturaRESUMO
Bacterial survival necessitates endurance of many types of antimicrobial compound. Many Gram-negative envelope stress responses, which must contend with an outer membrane and a dense periplasm containing the cell wall, have been associated with the status of protein folding, membrane homeostasis, and physiological functions such as efflux and the proton motive force (PMF). In this review, we discuss evidence that indicates an emerging role for Gram-negative envelope stress responses in enduring exposure to diverse antimicrobial substances, focusing on recent studies of the γ-proteobacterial Cpx envelope stress response.
Assuntos
Anti-Infecciosos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/fisiologia , Proteínas de Bactérias/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Meio Ambiente , Proteínas Quinases/fisiologia , Força Próton-Motriz/fisiologia , Transdução de Sinais , Estresse Fisiológico/fisiologiaRESUMO
During photosynthesis, photosynthetic electron transport generates a proton motive force (pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential (ΔΨ) and an osmotic component (ΔpH). Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments. However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin-like protein (AtBest), which is critical for pmf partitioning. While the ΔpH component was slightly reduced in atbest, the ΔΨ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching (NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. AtBest belongs to the bestrophin family proteins, which are believed to function as chloride (Cl- ) channels. Thus, our findings reveal an important Cl- channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine-tuning photosynthesis under fluctuating environmental conditions.