1,721,019 research outputs found
ATP Binding Causes a Conformational Change in the γ Subunit of the Escherichia coli F1ATPase Which Is Reversed on Bond Cleavage
No full textATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the subunit at position 106 [Turina, P.,& Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465–13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP•PNP and by rapid measurements using the slowly cleavable ATPS.The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATPS as substrate. With ATPS, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k+2 and k−2. The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATPS were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATPS binding causes the fluorescence enhancement of CM bound to the γ subunit and that this conformational change is reversed upon bond cleavage to yield ADP•Pi or ADP•PiS in catalytic sites. © 1994, American Chemical Society. All rights reserved
Structural changes during ATP hydrolysis activity of the ATP synthase from Escherichia coli as revealed by fluorescent probes
No full textF1F0-ATPase complexes undergo several changes in their tertiary and quaternary structure during their functioning. As a possible way to detect some of these different conformations during their activity, an environment-sensitive fluorescence probe was bound to cysteine residues, introduced by site-directed mutagenesis, in the γ subunit of the Escherichia coli enzyme. Fluorescence changes and ATP hydrolysis rates were compared under various conditions in F1 and in reconstituted F1F0. The results are discussed in terms of possible modes of operation of the ATP synthases
Influence of the transmembrane electrochemical proton gradient on catalysis and regulation of the H+-ATP synthase from Rhodobacter capsulatus
No full textFor a possible elucidation of the single steps of the reaction cycle it is essential to distinguish between regulatory and catalytic events. In this work we have investigated the effect of ΔμH+ on the activation and the rate of ATP synthesis of the H+-ATP synthase from Rhodobacter capsulatus. A gradient ΔμH+ was generated across the membrane of the chromatophores by subjecting them to acid-base transitions and to K+/valinomycin-induced diffusion potentials. The osmotic component ΔpH was varied by varying the pH of the acidic stage (internal pH) by constant external pH, and the electrical component ΔΦ was varied by varying the internal concentration of K+. The diffusion potentials generated were evaluated using the Goldman equation. The main advantages of this technique are the quantitative definition of ΔμH+ and the possibility of varying its chemical and electrical components independently. A rapid-mixing quenched-flow apparatus was used to measure the initial rate of ATP synthesis. The relative increase in the uncoupled rate of ATP hydrolysis after a ΔμH+ pulse was considered proportional to the degree of activation of the enzyme. The two components of ΔμH+ were found to be kinetically equivalent to driving ATP synthesis. However, in the same range of values, the electrical component was much more effective than the osmotic component in activating the enzyme. Taken together, these results raise the interesting possibility that well-tuned regulatory mechanisms may intervene to compensate a different kinetic effect of ΔΦ and ΔpH on the reaction cycle. © 1994
ATP hydrolysis-driven structural changes in the γ-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues
No full textFour mutants of the Escherichia coli F1ATPase, γS8-C, γT106-C, γS179- C, and γV286-C, which have a cysteine introduced at different sites in the γ-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue. With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored. The results obtained with the mutants γS8-C and γT106-C are similar. In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP. The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching. The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the γ- subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites. No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites. The fluorescence changes observed in mutants γS8-C and γT106-C were not seen in subunit ε-free E. coli F1ATPase, although such enzyme preparations are highly active ATPases. We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling
A point mutation in the ATP synthase of Rhodobacter capsulatus results in differential contributions of ΔpH and Δφ in driving the ATP synthesis reaction
No full textThe interface between the c-subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Δφ) at low transmembrane pH difference (ΔpH). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0, and the mechanism determining the relative contribution of ΔpH and Δφ in driving ATP synthesis
ATP synthesis in chromatophores driven by artificially induced ion gradients
No full textAn electrochemical potential difference for protons (μH+) across the membrane of bacterial chromatophores was induced by an artificially generated pH difference (pH) and a K+/valinomycin diffusion potential, φ. The initial rate of ATP synthesis was measured with a rapid‐mixing quenched‐flow apparatus in the time range between 70 ms and 30 s after the acid–base transition. The rate of ATP synthesis depends exponentially on pH;. Increasing diffusion potentials shift the pH dependency to lower pH values. Diffusion potentials were calculated from the Goldman equation. Using estimated permeability coefficients, the rate of ATP synthesis depends only on the electrochemical potential difference of protons irrespective of the relative contribution of pH and φ. Copyright © 1991, Wiley Blackwell. All rights reserve
The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force
No full textThe ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K+/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Δμ(H+) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Δμ(H+) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes
H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes
No full textThe H+/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H+-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pHin, could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid-base transition at different [ATP]/([ADP][Pi]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][Pi]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δμ̃H+ (eq), resulted in H+/ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05
ThermoScan: Semi-automatic Identification of Protein Stability Data From PubMed
Full text linkDuring the last years, the increasing number of DNA sequencing and protein mutagenesis studies has generated a large amount of variation data published in the biomedical literature. The collection of such data has been essential for the development and assessment of tools predicting the impact of protein variants at functional and structural levels. Nevertheless, the collection of manually curated data from literature is a highly time consuming and costly process that requires domain experts. In particular, the development of methods for predicting the effect of amino acid variants on protein stability relies on the thermodynamic data extracted from literature. In the past, such data were deposited in the ProTherm database, which however is no longer maintained since 2013. For facilitating the collection of protein thermodynamic data from literature, we developed the semi-automatic tool ThermoScan. ThermoScan is a text mining approach for the identification of relevant thermodynamic data on protein stability from full-text articles. The method relies on a regular expression searching for groups of words, including the most common conceptual words appearing in experimental studies on protein stability, several thermodynamic variables, and their units of measure. ThermoScan analyzes full-text articles from the PubMed Central Open Access subset and calculates an empiric score that allows the identification of manuscripts reporting thermodynamic data on protein stability. The method was optimized on a set of publications included in the ProTherm database, and tested on a new curated set of articles, manually selected for presence of thermodynamic data. The results show that ThermoScan returns accurate predictions and outperforms recently developed text-mining algorithms based on the analysis of publication abstracts. Availability: The ThermoScan server is freely accessible online at https://folding.biofold.org/thermoscan. The ThermoScan python code and the Google Chrome extension for submitting visualized PMC web pages to the ThermoScan server are available at https://github.com/biofold/ThermoScan
Regulation of Cytochrome c Oxidase by Interaction of ATP at Two Binding Sites, One on Subunit VIa
No full textCytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit Via had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit Via was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit Via in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) and 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit Via. Labeling of both tissue specific isoforms of bovine cytochrome c oxidase in subunit Via indicated that the ATP binding site is conserved in the subunit from different species as well as in different isoforms. Since the C-terminal part of subunit Via, which is located in the intermembrane space, is much more strongly conserved than the N-terminal part of the polypeptide, the labeling results suggest a common ATP binding site located at the C-terminal part of the polypeptide. Taken together, these observations support a regulatory role for subunit Via of cytochrome c oxidase in which this subunit monitors the concentration of ATP in the intermembrane space and inhibits the enzyme activity at physiological concentrations of ATP. © 1994, American Chemical Society. All rights reserved
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