Etude des polyphénols du houblon, de l'orge et de la bière en relation avec la stabilité physico-chimique de la bière

Thèse de doctorat -- Université catholique de Louvain, 196

A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase

A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from <i>Rhodopseudomonas palustris</i> as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively

Development of a range of fluorescent reagentless biosensors for ATP, based on malonyl-coenzyme A synthetase

<div><p>The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from <i>Rhodopseudomonas palustris</i>. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent’s 6 μM to 123 μM and 42 μM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 μM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 μM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present.</p></div

Fluorescence change and affinity for ATP binding to variants of Rho-MatB.

<p>Fluorescence change and affinity for ATP binding to variants of Rho-MatB.</p

Nucleotide affinity for variants of Rho-MatB.

<p>Titration of ATP (circles) and ADP (triangles) to (A) 0.5 µM Rho-MatB T167A, (B) 0.5 μM Rho-MatB T303A; (C) 0.5 μM Rho-MatB S170A. Measurements are shown for one representative experiment and were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 20°C. Excitation was at 553 nm, emission at 571 nm. The dissociation constants and fluorescence ratios were obtained using a quadratic binding equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#sec002" target="_blank">Methods</a>) and are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.t002" target="_blank">Table 2</a>. The drop lines are to indicate the dissociation constants. Data are normalized to the intensity in the absence of nucleotide.</p

Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.

<p>Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.</p

Test assay, measurement of the <i>K</i>_m of ADP with pyruvate kinase using T303A variant ATP biosensor.

<p>The assay was run in multiwell format in 50 mM HEPES buffer pH 7.2, 100 mM KCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 25°C. The assay components were 500 μM phosphoenolpyruvate, 0.005 unit ml^-1 pyruvate kinase, 1 μM T303A rho-MatB and the micromolar concentrations of ADP shown on the inset panel for an illustrative set of traces. The data are shown as ATP production rates, using calibrations done at 0–20 μM ATP at 0 μM ADP and with ADP to make 500 μM total nucleotide concentration. Intermediate calibrations were by interpolation. The average (with standard errors) are plotted for four independent measurements, giving <i>K</i>_m of 56 ± 4 μM.</p

Association kinetics of ATP binding to Rho-MatB variants.

<p>Fluorescence time courses were measured by rapidly mixing 0.25 µM Rho-MatB with different concentrations of ATP in large excess (micromolar concentrations shown). Measurements for one representative dataset are shown and were obtained in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 20°C. Time courses were obtained for two different time scales to show fast and slow phases. Slow phases are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.s003" target="_blank">S3 Fig</a>. Note that the dead time of the stopped-flow instrument (the time taken for the mixed solution to reach the fluorescence observation chamber) is ~2 ms, so that the traces of the fast phase only show changes from that time. (A) Example traces for Rho-MatB T167A, T303A and S170A variants. (B) The average, observed rate constants are shown with standard errors. The fast phases were fit to single exponentials to give rate constants (<i>k</i>_obs), increasing linearly with ATP concentration. “+AMP” is a set of measurements with the parent Rho-MatB in the presence of 500 µM AMP. (C) Conformational selection model for binding derived for Rho-MatB: step 1 is a conformation change of the apoprotein, this is followed by ATP binding. The star indicates the high fluorescence state. Pseudo-first order conditions used for the kinetic measurements give <i>k</i>_obs = <i>k</i>₊₂[ATP] + <i>k</i>_-2: the slope (second order rate constant for association, <i>k</i>₊₂) and intercept (dissociation rate constant, <i>k</i>_-2) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.t002" target="_blank">Table 2</a>.</p