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Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb²⁺.

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

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Time-tracing curves of conformational changes by measuring absorbance change at 303 nm for (a) Na+/Pb2+ and (b) K+/Pb2+ exchanges. PS2.M (5 µM) was annealed in the buffer (10 mM/Tris, pH 6.1) containing 50 mM of Na+ or K+ and then mixed with Pb(NO3)2 (50 µM) at 25°C. Each profile was fitted to a three-exponential function. For the Na+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.0037 ± 0.0001 s, τ2 = 0.15 ± 0.001 s and τ3 = 2.1 ± 0.01 s. For the K+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.23 ± 0.007 s, τ2 = 1.88 ± 0.03 s and τ3 = 17.6 ± 0.2 s. The residual plots indicate the deviation of the experimental and fitted absorbance changes.
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gkr1310-F6: Time-tracing curves of conformational changes by measuring absorbance change at 303 nm for (a) Na+/Pb2+ and (b) K+/Pb2+ exchanges. PS2.M (5 µM) was annealed in the buffer (10 mM/Tris, pH 6.1) containing 50 mM of Na+ or K+ and then mixed with Pb(NO3)2 (50 µM) at 25°C. Each profile was fitted to a three-exponential function. For the Na+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.0037 ± 0.0001 s, τ2 = 0.15 ± 0.001 s and τ3 = 2.1 ± 0.01 s. For the K+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.23 ± 0.007 s, τ2 = 1.88 ± 0.03 s and τ3 = 17.6 ± 0.2 s. The residual plots indicate the deviation of the experimental and fitted absorbance changes.

Mentions: Na+ and K+ ions are the major intracellular metal cations, which are also able to induce folding of G-quadruplex. We find that Pb2+ ions can not only assemble G-rich DNA segments into a G-quadruplex, but also ‘switch’ quadruplex structures by readily displacing K+ or Na+ ions from the quadruplexes. To determine whether the effect of Pb2+ ions on the structural rearrangement is synergetic or competitive, we investigated the conformational switches in the G-quadruplexes of PS2.M induced by Na+ or K+ ions in the presence of Pb2+ ions. Figure 6 shows the progress curves for the Na+→Pb2+ and K+→Pb2+ exchanges measured using UV stopped-flow spectrophotometry at 303 nm. A slight increase in ΔA was observed after the addition of Pb(NO3)2, and the distinctly different spectra reveal that Pb2+ ions ‘switch’ further structural transitions of the Na+- or K+-induced G-quadruplex.Figure 6.


Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb²⁺.

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Time-tracing curves of conformational changes by measuring absorbance change at 303 nm for (a) Na+/Pb2+ and (b) K+/Pb2+ exchanges. PS2.M (5 µM) was annealed in the buffer (10 mM/Tris, pH 6.1) containing 50 mM of Na+ or K+ and then mixed with Pb(NO3)2 (50 µM) at 25°C. Each profile was fitted to a three-exponential function. For the Na+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.0037 ± 0.0001 s, τ2 = 0.15 ± 0.001 s and τ3 = 2.1 ± 0.01 s. For the K+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.23 ± 0.007 s, τ2 = 1.88 ± 0.03 s and τ3 = 17.6 ± 0.2 s. The residual plots indicate the deviation of the experimental and fitted absorbance changes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351173&req=5

gkr1310-F6: Time-tracing curves of conformational changes by measuring absorbance change at 303 nm for (a) Na+/Pb2+ and (b) K+/Pb2+ exchanges. PS2.M (5 µM) was annealed in the buffer (10 mM/Tris, pH 6.1) containing 50 mM of Na+ or K+ and then mixed with Pb(NO3)2 (50 µM) at 25°C. Each profile was fitted to a three-exponential function. For the Na+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.0037 ± 0.0001 s, τ2 = 0.15 ± 0.001 s and τ3 = 2.1 ± 0.01 s. For the K+/Pb2+ exchange, the optimized relaxation times are τ1 = 0.23 ± 0.007 s, τ2 = 1.88 ± 0.03 s and τ3 = 17.6 ± 0.2 s. The residual plots indicate the deviation of the experimental and fitted absorbance changes.
Mentions: Na+ and K+ ions are the major intracellular metal cations, which are also able to induce folding of G-quadruplex. We find that Pb2+ ions can not only assemble G-rich DNA segments into a G-quadruplex, but also ‘switch’ quadruplex structures by readily displacing K+ or Na+ ions from the quadruplexes. To determine whether the effect of Pb2+ ions on the structural rearrangement is synergetic or competitive, we investigated the conformational switches in the G-quadruplexes of PS2.M induced by Na+ or K+ ions in the presence of Pb2+ ions. Figure 6 shows the progress curves for the Na+→Pb2+ and K+→Pb2+ exchanges measured using UV stopped-flow spectrophotometry at 303 nm. A slight increase in ΔA was observed after the addition of Pb(NO3)2, and the distinctly different spectra reveal that Pb2+ ions ‘switch’ further structural transitions of the Na+- or K+-induced G-quadruplex.Figure 6.

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

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