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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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Representative kinetics (kon) measurements from titrations in either 0.5 mM MgCl2 or 150 mM KCl at 20°C (titrations are the same as those in Figures 2 and 3). (A) Selected deconvolved energies (Edc) and fits for peaks 2, 7 and 12 for titration performed in 0.5 mM MgCl2, 20°C. Bold red lines represent target function description of the data using best-fit parameters over the time range used in fitting; extrapolations of these functions are displayed in thin red lines. Peaks 7 and 12 are arbitrarily offset from 0 to aid visualization. (B) Representative peaks and fit functions for titration in 150 mM KCl, 20°C. (C) Corresponding complete population of kon values from sampled titrations. Solid squares, KCl data; Open circles, MgCl2 data.
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gkr894-F4: Representative kinetics (kon) measurements from titrations in either 0.5 mM MgCl2 or 150 mM KCl at 20°C (titrations are the same as those in Figures 2 and 3). (A) Selected deconvolved energies (Edc) and fits for peaks 2, 7 and 12 for titration performed in 0.5 mM MgCl2, 20°C. Bold red lines represent target function description of the data using best-fit parameters over the time range used in fitting; extrapolations of these functions are displayed in thin red lines. Peaks 7 and 12 are arbitrarily offset from 0 to aid visualization. (B) Representative peaks and fit functions for titration in 150 mM KCl, 20°C. (C) Corresponding complete population of kon values from sampled titrations. Solid squares, KCl data; Open circles, MgCl2 data.

Mentions: Representative kinetic analyses of the titrations in Figures 2 and 3 are displayed in Figure 4. Figure 4A plots the deconvolved power trace and respective fitted curves for injections 2, 7 and 12 from the representative titration collected in 0.5 mM MgCl2, 20°C, and a similar sampling of peaks and fits from the 150 mM KCl, 20°C dataset is displayed in Panel B. The resulting forward rate constants, kon, are plotted for each injection from the first half of each titration (all injections such that [TT]total > [RR]total) in Figure 4C. As is visible in the raw data (Figure 2A), the observed rate of association decreases appreciably with each subsequent injection over the course of the first half of the titration, reflecting the decreasing concentration of unbound RR. Because kon is a microscopic rate constant, it is independent of the RNA concentration (Figure 4C); the weighted-average kon for these experiments are 860 (±50) M−1 s−1 in MgCl2 and 2180 (±240) M−1 s−1 in KCl. The faster association of TT–RR in KCl versus MgCl2 solution at ambient temperatures and respective salt concentrations eliciting similar stabilities is consistent with extant RNA folding literature (22–24).Figure 4.


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)

Representative kinetics (kon) measurements from titrations in either 0.5 mM MgCl2 or 150 mM KCl at 20°C (titrations are the same as those in Figures 2 and 3). (A) Selected deconvolved energies (Edc) and fits for peaks 2, 7 and 12 for titration performed in 0.5 mM MgCl2, 20°C. Bold red lines represent target function description of the data using best-fit parameters over the time range used in fitting; extrapolations of these functions are displayed in thin red lines. Peaks 7 and 12 are arbitrarily offset from 0 to aid visualization. (B) Representative peaks and fit functions for titration in 150 mM KCl, 20°C. (C) Corresponding complete population of kon values from sampled titrations. Solid squares, KCl data; Open circles, MgCl2 data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr894-F4: Representative kinetics (kon) measurements from titrations in either 0.5 mM MgCl2 or 150 mM KCl at 20°C (titrations are the same as those in Figures 2 and 3). (A) Selected deconvolved energies (Edc) and fits for peaks 2, 7 and 12 for titration performed in 0.5 mM MgCl2, 20°C. Bold red lines represent target function description of the data using best-fit parameters over the time range used in fitting; extrapolations of these functions are displayed in thin red lines. Peaks 7 and 12 are arbitrarily offset from 0 to aid visualization. (B) Representative peaks and fit functions for titration in 150 mM KCl, 20°C. (C) Corresponding complete population of kon values from sampled titrations. Solid squares, KCl data; Open circles, MgCl2 data.
Mentions: Representative kinetic analyses of the titrations in Figures 2 and 3 are displayed in Figure 4. Figure 4A plots the deconvolved power trace and respective fitted curves for injections 2, 7 and 12 from the representative titration collected in 0.5 mM MgCl2, 20°C, and a similar sampling of peaks and fits from the 150 mM KCl, 20°C dataset is displayed in Panel B. The resulting forward rate constants, kon, are plotted for each injection from the first half of each titration (all injections such that [TT]total > [RR]total) in Figure 4C. As is visible in the raw data (Figure 2A), the observed rate of association decreases appreciably with each subsequent injection over the course of the first half of the titration, reflecting the decreasing concentration of unbound RR. Because kon is a microscopic rate constant, it is independent of the RNA concentration (Figure 4C); the weighted-average kon for these experiments are 860 (±50) M−1 s−1 in MgCl2 and 2180 (±240) M−1 s−1 in KCl. The faster association of TT–RR in KCl versus MgCl2 solution at ambient temperatures and respective salt concentrations eliciting similar stabilities is consistent with extant RNA folding literature (22–24).Figure 4.

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.

Show MeSH
Related in: MedlinePlus