Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59.The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA.We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.     

Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.     

Conformation of hyaluronate in neutral and alkaline solutions

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

Spectral manifestations of the effect of Zn2+ ions on DNA complexes with distmycin

Effect of low concentrations of metal bivalent ions on DNA-distamycin complexes was studied. It has been found that when adding small quantity of Zn2- ions the DNA-distamycin complex changes its cooperative properties. A small amount of heavy metal ions incorporated in DNA changes the DNA property to form complexes capable of stimulating the formation of structures of high organization. Thus under the effect of metal small additions both the structure of cellular processes and their regulation are irreversibly simplified, the phase of rest characterized by the maximal number of organization levels of the nuclear apparatus not being reached.     

Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases.

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.     

Conformation of hyaluronate in neutral and alkaline solutions.

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

Raman spectroscopic study of the effects of Ca2+, Mg2+, Zn2+, and Cd2+ ions on calf thymus DNA: binding sites and conformational changes

The interaction of Mg2+, Ca2+, Zn2+, and Cd2+ with calf thymus DNA has been investigated by Raman spectroscopy. These spectra reveal that all of these ions, and particularly Zn2+, bind to phosphate groups of DNA, causing a slight structural change in the polynucleotide at very small metal: DNA (P) concentration ratio (ca. 1:30). This results in increased base-stacking interactions, with negligible change of the B conformation of DNA. Contrary to Zn2+ and Cd2+, which interact extensively with the nucleic bases (particularly at the N7 position of guanine), the alkaline-earth metal ions are bound almost exclusively to the phosphate groups. The affinity of both the Zn2+ and Cd2+ ions for G.C base pairs is comparable, but the Cd2+ ions interact more extensively with A.T pairs than Zn2+ ions. Interstrand cross-linking through the N3 atom of cytosine is suggested in the presence of Zn2+, but not Cd2+.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

Interactions of metallic ions with DNA. V. DNA renaturation mechanism in the presence of Cu 2+

New experimental data were obtained by means of circular dichroism, melting, renaturation, and kinetic experiments, upon Cu²⁺ binding to DNA, poly dAT, and poly dGdC. They enable us to propose a model of binding giving a satisfactory explanation to all of the data found in the literature. Two types of binding sites are proposed: (a) a “sandwich” of Cu²⁺ between two adjacent G-C pairs giving a charge-transfer complex, and (b) a chelate between a phosphate group and a nitrogen atom of the bases (N₇ of guanine and N₃ of cytosine at room temperature, N₃ of adenine after thermal opening of A-T pair). Type (a) stabilizes the helix and keeps the two strands linked. Type (b) destabilizes the helix and explains why the kinetic rate of renaturation is the same as that of copper release.     

Application of polyelectrolyte theories for analysis of DNA melting in the presence of Na+ and Mg2+ ions.

Numerical calculations, using Poisson-Boltzmann (PB) and counterion condensation (CC) polyelectrolyte theories, of the electrostatic free energy difference, DeltaGel, between single-stranded (coil) and double-helical DNA have been performed for solutions of NaDNA + NaCl with and without added MgCl2. Calculations have been made for conditions relevant to systems where experimental values of helix coil transition temperature (Tm) and other thermodynamic quantities have been measured. Comparison with experimental data has been possible by invoking values of Tm for solutions containing NaCl salt only. Resulting theoretical values of enthalpy, entropy, and heat capacity (for NaCl salt-containing solutions) and of Tm as a function of NaCl concentration in NaCl + MgCl2 solutions have thus been obtained. Qualitative and, to a large extent, quantitative reproduction of the experimental Tm, DeltaHm, DeltaSm, and DeltaCp values have been found from the results of polyelectrolyte theories. However, the quantitative resemblance of experimental data is considerably better for PB theory as compared to the CC model. Furthermore, some rather implausible qualitative conclusions are obtained within the CC results for DNA melting in NaCl + MgCl2 solutions. Our results argue in favor of the Poisson-Boltzmann theory, as compared to the counterion condensation theory.     

Denaturation of DNA in the presence of chromous (Cr2+) ions

The effect of Cr²⁺ ions on the T_m (melting temperature) of DNA has been investigated under appropriate conditions for the stabilization of DNA by Mg²⁺ ions. A significant lowering of T_m, analogous to that observed for Cu²⁺ under normal conditions, was found, for Cr²⁺ at pH = 4.2 and [Mg²⁺] = 5.3 mol per mole of DNA base pair. Cu²⁺ also lowers T_m under similar conditions. The similarity of the effects of Cr²⁺ and Cu²⁺ under comparable conditions may be related to similarities in their coordination properties. It is proposed that Cr²⁺ and Cu²⁺ ions facilitate denaturation by holding together groups on the DNA chains in such a manner that base pairing and base stacking are inhibited. Comparative results for Cr³⁺ and Co²⁺ are also given for these low pH/Mg²⁺ ion conditions.     

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

Cooperative conformational change and aggregation of concanavalin A in alkaline solutions

Three properties, the binding activity to Sephadex G-75, conformation, and the extent of aggregation, of concanvalin A. (con A) in alkaline pH solutions were examined with special attention to the time course and their time-independent final values. Highly cooperative conformational changes among four subunits were suggested which were coupled either with protonation in the case of demetallized con A or with metal binding in the case of metal-liganded con A. Midpoints of the conversions of the metal-liganded con A were about pH 8.8, 9.1 and 9.1 with respect to the activity, the conformational change and the aggregation, respectively. These values were about 1 pH higher than the corresponding values of demetallized con A: 7.9, 8.05 and 8.2. Each conversion took place in narrow pH ranges. The pH range for the loss of activity was found to be significantly lower than those of the other two. The aggregation was suggested not to be coupled with the conformational change. Dissociation into subunits did not take place indicating strong interactions among four subunits in the tetramer.     

The influence of Zn2+ ions on the tryptophan biosynthesis in plants

Zn²⁺ ions slightly enhance at low concentrations (0.01 μg ml^-1) the activity of tryptophan synthase obtained from the shoots of 14-day-old pea seedlings (Pisum sativum L.). On the contrary, high concentrations of Zn²⁺ (10 μg ml^-l) exert an inhibitory effect. The direct Zn²⁺ activation of tryptophan synthase, establishedin vitro with a partially purified enzyme preparation, is relatively low and obviously is not decisive from the point of view of tryptophan biosynthesis of the enzyme and thus they are participating in thein vivo experiments.