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Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry.

Vander Meulen KA, Butcher SE - Nucleic Acids Res. (2011)

Bottom Line: The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium.These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)).The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr Madison, WI 53706, USA. kvandermeulen@biochem.wisc.edu

ABSTRACT
A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop-receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH(‡)) and Eyring transition state entropies (ΔS(‡)). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH(25°C) = -41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH(‡) = -0.6 ± 0.5). These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

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Thermodynamics of TT–RR binding in MgCl2 and KCl solutions. (A) Representative titration comparison in conditions eliciting comparable complex stabilities. Open circles, 0.5 mM MgCl2, 20°C, titrant [TT] = 229 µM, starting cell [RR] = 10 µM. Solid squares, 150 mM KCl, 20°C, titrant [TT] = 145 µM, starting cell [RR] = 10 µM. (B) Left, linear fit-determined maximum likelihood ΔG25C, ΔH25C and TΔS25C in 0.3 (red), 0.5 (green), 0.7 (blue) and 1.0 mM (black) MgCl2. Right, ΔG25C, ΔH25C and TΔS25C in 100 (red), 150 (green) and 200 (blue) mM KCl.
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gkr894-F3: Thermodynamics of TT–RR binding in MgCl2 and KCl solutions. (A) Representative titration comparison in conditions eliciting comparable complex stabilities. Open circles, 0.5 mM MgCl2, 20°C, titrant [TT] = 229 µM, starting cell [RR] = 10 µM. Solid squares, 150 mM KCl, 20°C, titrant [TT] = 145 µM, starting cell [RR] = 10 µM. (B) Left, linear fit-determined maximum likelihood ΔG25C, ΔH25C and TΔS25C in 0.3 (red), 0.5 (green), 0.7 (blue) and 1.0 mM (black) MgCl2. Right, ΔG25C, ΔH25C and TΔS25C in 100 (red), 150 (green) and 200 (blue) mM KCl.

Mentions: Analysis of TT–RR binding data reveals strikingly different thermodynamic profiles for this helical packing event in MgCl2 and KCl solutions (Figure 3). Representative experiments performed at 20°C in either 0.5 mM MgCl2 or 150 mM KCl demonstrate that, while binding is enthalpy-driven in both salts, in KCl it is significantly more exothermic (for these conditions, on average ΔΔH = −21.7 (±3.7) kcal/mol) and less favorable entropically (Δ(−TΔS) = +22.4 (±3.8) kcal/mol) (Figure 3A). The differences in these terms represent contributions to ΔG that are nearly three times greater than the overall stabilities.Figure 3.


Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry.

Vander Meulen KA, Butcher SE - Nucleic Acids Res. (2011)

Thermodynamics of TT–RR binding in MgCl2 and KCl solutions. (A) Representative titration comparison in conditions eliciting comparable complex stabilities. Open circles, 0.5 mM MgCl2, 20°C, titrant [TT] = 229 µM, starting cell [RR] = 10 µM. Solid squares, 150 mM KCl, 20°C, titrant [TT] = 145 µM, starting cell [RR] = 10 µM. (B) Left, linear fit-determined maximum likelihood ΔG25C, ΔH25C and TΔS25C in 0.3 (red), 0.5 (green), 0.7 (blue) and 1.0 mM (black) MgCl2. Right, ΔG25C, ΔH25C and TΔS25C in 100 (red), 150 (green) and 200 (blue) mM KCl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300012&req=5

gkr894-F3: Thermodynamics of TT–RR binding in MgCl2 and KCl solutions. (A) Representative titration comparison in conditions eliciting comparable complex stabilities. Open circles, 0.5 mM MgCl2, 20°C, titrant [TT] = 229 µM, starting cell [RR] = 10 µM. Solid squares, 150 mM KCl, 20°C, titrant [TT] = 145 µM, starting cell [RR] = 10 µM. (B) Left, linear fit-determined maximum likelihood ΔG25C, ΔH25C and TΔS25C in 0.3 (red), 0.5 (green), 0.7 (blue) and 1.0 mM (black) MgCl2. Right, ΔG25C, ΔH25C and TΔS25C in 100 (red), 150 (green) and 200 (blue) mM KCl.
Mentions: Analysis of TT–RR binding data reveals strikingly different thermodynamic profiles for this helical packing event in MgCl2 and KCl solutions (Figure 3). Representative experiments performed at 20°C in either 0.5 mM MgCl2 or 150 mM KCl demonstrate that, while binding is enthalpy-driven in both salts, in KCl it is significantly more exothermic (for these conditions, on average ΔΔH = −21.7 (±3.7) kcal/mol) and less favorable entropically (Δ(−TΔS) = +22.4 (±3.8) kcal/mol) (Figure 3A). The differences in these terms represent contributions to ΔG that are nearly three times greater than the overall stabilities.Figure 3.

Bottom Line: The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium.These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)).The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr Madison, WI 53706, USA. kvandermeulen@biochem.wisc.edu

ABSTRACT
A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop-receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH(‡)) and Eyring transition state entropies (ΔS(‡)). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH(25°C) = -41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH(‡) = -0.6 ± 0.5). These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

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