Kinetics Of Fluorescence Changes Research Articles

Characterization of the Binding Properties of Ten Aptamers Using the Intrinsic Fluorescence of Oxytetracycline.

Tetracyclines are a class of commonly used four-ringed antibiotics. A series of DNA aptamers were recently obtained using the capture-SELEX (systematic evolution of ligands by exponential enrichment) method to bind to oxytetracycline, and one of the aptamers can bind to a few other tetracycline antibiotics as well. Upon binding to the aptamers, the intrinsic fluorescence of tetracycline antibiotics can be enhanced. At least 10 different DNA aptamers were isolated from the previous selection experiment. In this work, a systematic characterization of these ten aptamers was performed. Each of these aptamers shows a different degree of fluorescence enhancement ranging from around 1-fold to over 20-fold. Fluorescence enhancement was boosted in the presence of Mg2+. Isothermal titration calorimetry (ITC) studies were done and showed a great variety in dissociation constant (Kd) from 62 nM to 1.6 μM. Steady-state fluorescence spectroscopy and fluorescence lifetime studies showed a correlation between fluorescence lifetime and degree of fluorescence enhancement. A few aptamers showed slow binding kinetics, although no correlation was found between the kinetics of fluorescence change and degree of fluorescence enhancement. This is the first study of ten different aptamers for the same target, providing fundamental insights into aptamer binding and bioanalytical applications.

A simple routine for quantitative analysis of light and dark kinetics of photochemical and non-photochemical quenching of chlorophyll fluorescence in intact leaves.

Paper describes principles and application of a novel routine that enables the quantitative analysis of the photochemical O–J phase of the variable fluorescence F v associated with the reversible photo-reduction of the secondary electron acceptor QA of photosystem II (PSII) in algae and intact leaves. The kinetic parameters that determine the variable fluorescence F PP(t) associated with the release of photochemical quenching are estimated from 10 µs time-resolved light-on and light-off responses of F v induced by two subsequent light pulses of 0.25 (default) and 1000 ms duration, respectively. Application of these pulses allows estimations of (i) the actual value of the rate constants k L and k AB of the light excitation (photoreduction of QA) and of the dark re-oxidation of photoreduced QA ({text{Q}}_{text{A}}^{ - }), respectively, (ii) the actual maximal normalized variable fluorescence [nF v] associated with 100 % photoreduction of QA of open RCs, and (iii) the actual size β of RCs in which the re-oxidation of {text{Q}}_{text{A}}^{ - } is largely suppressed (QB-nonreducing RC with k AB ~ 0). The rate constants of the dark reversion of Fv associated with the release of photo-electrochemical quenching F PE and photo-electric stimulation F CET in the successive J–I and I–P parts of the thermal phase are in the range of (100 ms)−1 and (1 s)−1, respectively. The kinetics of fluorescence changes during and after the I–P phase are given special attention in relation to the hypothesis on the involvement of a Δµ H+-dependent effect during this phase and thereafter. Paper closes with author’s personal view on the demands that should be fulfilled for chlorophyll fluorescence methods being a correct and unchallenged signature of photosynthesis in algae and plants.

Ultrafast Genetically Encoded Calcium Indicators for Visualizing Calcium Flux and Action Potentials

Genetically encoded calmodulin-based calcium probes (GCaMPs) have become the reporters of choice for visualising the calcium flux associated with action potentials in vivo. A major limitation of currently available GCaMPs is the slow kinetics of fluorescence changes induced by calcium association and dissociation. We have addressed this issue by generating a series of mutants of GCaMP3 in the calcium binding sites of calmodulin alone1 and in combination with mutations in the RS20 target peptide sequence2 with the view of lowering the affinity for calcium and accelerating the calcium response kinetics. The calcium association kinetics for the resulting GCaMP3 EF-hand and peptide mutants were highly cooperative and characterized by a rate limiting conformational change. Fluorescence changes on calcium association were up to 7-fold faster compared to GCaMP3. Calcium dissociation rates were up to 60-fold faster than GCaMP3 and 25-fold faster than the newly developed GCaMP6 fast (GCaMP6f). Dissociation constants (Kd) for calcium were in the μM range with Hill coefficients from 2 to 5. Two-photon cross-sections of mutants were comparable to GCaMP3. Fluorescence responses of mutated GCaMP3s to calcium transients in endothelial cells were similar to those seen with small molecule indicators. The principles employed proved to accelerate the calcium kinetics of GCaMP3 and can be applied to the new generations of GCaMPs to generate low affinity probes.1Jama A et al. JBC, 2011, 286:12308-12316.2Torok K and Trentham DR. Biochemistry, 1994, 33:12807-12820.This work is funded by the Wellcome Trust grant number 094385/Z/10/Z to KT.

A liposomal fluorescence assay to study permeation kinetics of drug-like weak bases across the lipid bilayer

Lipid bilayer permeation is considered the major route for in vivo barrier passage of drugs. Despite this fact, no technique is currently available to measure the kinetics of permeation across a single lipid bilayer of structurally unrelated drug-like solutes. We developed a liposomal fluorescence assay capable to determine permeation kinetics of basic drug-like solutes across lipid bilayers. The assay is based on the hypothesis that permeation of a weak base along a concentration gradient results in net proton release at the cis-side and net proton capture at the trans-side of the bilayer. The resulting pH changes were monitored with pH-sensitive fluorophores: Test compounds were incubated with liposomes containing a pH-sensitive fluorophore at the bilayer surfaces or in the aqueous lumen and fluorescence changes were monitored with a stopped‐flow apparatus in solution or by total internal reflection fluorescence microscopy with surface-captured liposomes on a microfluidic platform. Incubation with lipophilic basic drugs resulted in the expected fluorescence changes while incubation with compounds without basic functionality or high polarity did not affect fluorescence. Kinetics of fluorescence changes followed bi-exponential functions. Logarithmic permeation coefficients (logPermapp) determined in solution and by microfluidics technology showed a good correlation (r2=0.94, n=7) and logPermapp increased with increasing lipophilicity. Neither diffusion in the aqueous phase nor partitioning into the bilayer was rate-limiting. PEGylation of 2% of the liposomal lipids reduced Permapp by a factor ~300. In conclusion, the presented liposomal fluorescence assay is capable to determine permeation kinetics of weak basic drug-like solutes across lipid bilayers. The method is adaptable to microfluidics technology for high-throughput measurements and can potentially be modified to work for weak acid solutes.

The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides

In its forward direction, transhydrogenase couples the reduction of NADP + by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a “cyclic reaction” catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP + and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD + and NADH. Extrapolating to the intact enzyme this binding to the “wrong” site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.

Measuring The Contribution Of S4 Charges On Gating Currents Of A Sodium Channel

Upon membrane depolarization, conformational changes in the S4 voltage sensors results in the opening of the voltage-gated sodium channel. The movement of the positively charged residues on the four distinct voltage-sensors in the membrane electric field generates a measurable transient current referred to as the gating current. The ON gating currents of the Na+ channel has two kinetic components and the fast component correlates well with the kinetics of fluorescence changes of probes attached to the voltage sensors of domains I, II, and III. The slow component of the gating current, however, matches with the fluorescence kinetics of probes on domain IV voltage-sensor (Chanda and Bezanilla JGP 2002 120; 629-45). In an attempt to specify the molecular origin of the slow and fast components of the gating current, we neutralized the first three charged residues (Q3 mutants) in each of the four voltage-sensing segments of the sodium channel. Our data supports the hypothesis that the voltage-sensor of domain IV primarily contributes to the slow component of the gating current and provides support to the notion that the movement of this voltage sensor is slower than the opening of the pore.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Microinjection in combination with microfluorimetry to study proton diffusion along phospholipid membranes

Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum(2)/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane.

Fluorometric Measurements of Intermolecular Distances between the α- and β-Subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.

On the advantages of using green light to study fluorescence yield changes in leaves

In photosynthetic chains, the kinetics of fluorescence yield depends on the photochemical rates at the level of both Photosystem I and II and thus on the absorption cross section of the photosynthetic units as well as on the coupling between light harvesting complexes and photosynthetic traps. A new set-up is described which, at variance with the commonly used set-ups, uses of a weakly absorbed light source (light-emitting diodes with maximum output at 520 nm) to excite the photosynthetic electron chain and probe the resulting fluorescence yield changes and their time course. This approach optimizes the homogeneity of the exciting light throughout the leaf and we show that this homogeneity narrows the distribution of the photochemical rates. Although the exciting light is weakly absorbed, the possibility to tune the intensity of the light emitting diodes allows one to reach photochemical rates ranging from 10 4 s − 1 to 0.25 s − 1 rendering experimentally accessible different functional regimes. The variations of the fluorescence yield induced by the photosynthetic activity are qualitatively and quantitatively discussed. When illuminating dark-adapted leaves by a weak light, the kinetics of fluorescence changes displays a pronounced plateau which precedes the fluorescence increase reflecting the full reduction of the plastoquinone pool. We ascribe this plateau to the time delay needed to reduce the photosystem I electron acceptors.

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Slow ligand-induced transitions in the allosteric phosphofructokinase from Escherichia coli

The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or effector, suggesting that the protein undergoes a conformational change. This fluorescent probe has been exploited to determine the rates of the structural transitions that occur upon ligand binding and that are responsible for the remarkable allosteric behavior of this enzyme. The kinetics of fluorescence changes measured after rapidly mixing phosphofructokinase with one of its ligands show the presence of several allosteric transitions with widely different rates, ranging from a few hundred s-1 to less than 0.1 s-1. The rate of each conformational change increases with the concentration of the ligand used to trigger it, suggesting that ligands induce a conformational change and do not displace a pre-existing equilibrium. The hypothesis that each ligand stabilizes a different conformational state of the protein is confirmed by the kinetics of displacement of one ligand by another: for instance, the binary complexes between phosphofructokinase and either its substrate, fructose-6-phosphate, or its allosteric activator, ADP, have the same low fluorescence and should be in the same active state, but they show different rates of conformational transition upon binding the inhibitor phosphoenolpyruvate. It appears that phosphofructokinase can exist in more than two states. Some conformational changes between these multiple states are slow enough to play an important role in the kinetics of the reaction catalyzed by phosphofructokinase, and could even explain part of its allosteric behavior. These results show that steady-state measurements are not sufficient to analyze the regulatory properties of E. coli phosphofructokinase.

Spectral characterization of environment-sensitive adducts of interleukin-1 beta.

We have determined the fluorescence properties of two covalently attached acrylodan derivatives of recombinant human interleukin-1 beta, namely the Cys-8 and Lys-103 adducts. The emission and excitation maxima indicated the presence of two operationally distinct conformers for each probe. The iodide quenching and the kinetics of fluorescence changes associated with guanidinium chloride-induced denaturation show that each covalent adduct exists both in hydrated and dehydrated environments. Furthermore, fluorescence changes associated with the binding of the adducts to a recombinant soluble murine receptor indicated that only the conformers with the label in the hydrophobic environment are competent toward the soluble murine interleukin receptor and that the hydrated and dehydrated conformers are in a dynamic equilibrium on the time scale of the binding experiments.

Purification and characterization of four monofluorescein cobra alpha-toxin derivatives.

We labeled cobra-alpha-toxin (Naja naja siamensis 3) with near stoichiometric quantities of fluorescein isothiocyanate. To reduce labeling of the hyperreactive N epsilon-lysine 23, the alpha-toxin was modified reversibly with citraconic anhydride before fluorescein labeling. The citraconic anhydride was later removed with strong acid, and four of the six possible monofluorescein alpha-toxin derivatives were isolated by isoelectric focusing on an immobilized pH gradient. Thermolysin digestion and subsequent high pressure liquid chromatography of the peptides yielded one dominant fluorescent peak from three of the isolated monofluorescein derivatives. Sequence analyses of these three fluorescent peaks indicated monofluorescein labeling at Lys-69, Lys-35, and Lys-49. Since one derivative (not identified by sequence analysis) displayed essentially identical chromatographic, spectroscopic, and binding properties as our previously identified monofluorescein-Lys-23 toxin (Johnson, D. A., and Taylor, P. (1982) J. Biol. Chem. 257, 5632-5636), we identified the site of labeling of this fourth derivative to be Lys-23. While only small differences were observed in the extinction maxima and molar extinction coefficients, the quantum yields of the isolated derivatives varied markedly and ranged between 0.18 and 0.41. Binding of monofluorescein-Lys-69, -Lys-35, -Lys-49, and -Lys-23 derivatives to the membrane-associated acetylcholine receptor from Torpedo californica was associated with -39, -26, -9, and +96% changes in fluorescence emission intensity, respectively. Based on analyses of the kinetics of fluorescence changes associated with receptor binding, the association and dissociation rate constants were measured. Relative to native cobra alpha-toxin, monofluorescein conjugation reduced the bimolecular association rate constants for binding to the receptor 13-33-fold. The dissociation rate binding rate constants were less affected and were reduced 0-5-fold.

Folding of yeast iso-1-AM cytochrome c.

We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group. The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein. The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride. The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing. The results are compared to those for the yeast isozyme, iso-2 cytochrome c. The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9).