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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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Eyring plot of both association (kon) and dissociation (koff) rate constants measured in MgCl2 and KCl solutions. Each symbol represents the average rate constant measurement from a single titration, and error bars represent the measurement's true uncertainty as described in Supplementary Material. Circles, kon; squares, koff. For both rate constants, closed symbols reflect data that were obtained from titrations where Ka was measured directly (1 × 105 M−1 < Ka < 2 × 107 M−1), while for open symbols, Ka > 2 × 107 M−1. Lines were generated using best-fit regression parameters. (A) MgCl2 data, (B) KCl data. Y- and X-axis scaling are identical in the two panels.
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gkr894-F5: Eyring plot of both association (kon) and dissociation (koff) rate constants measured in MgCl2 and KCl solutions. Each symbol represents the average rate constant measurement from a single titration, and error bars represent the measurement's true uncertainty as described in Supplementary Material. Circles, kon; squares, koff. For both rate constants, closed symbols reflect data that were obtained from titrations where Ka was measured directly (1 × 105 M−1 < Ka < 2 × 107 M−1), while for open symbols, Ka > 2 × 107 M−1. Lines were generated using best-fit regression parameters. (A) MgCl2 data, (B) KCl data. Y- and X-axis scaling are identical in the two panels.

Mentions: Figure 5 plots the logarithms of kon and koff against inverse temperature for all of the experiments in this work. In MgCl2 (Figure 5A), the slopes (−ΔH‡/R) for each salt concentration and both reaction directions are negative, indicating that ΔH‡ is large and positive for both association and dissociation. The full set of transition state theory thermodynamic quantities are listed in Table 4. is on average 8.1 (±0.5) kcal/mol in MgCl2 solutions. This is consistent with a previous measurement, which used an oligonucleotide linker to monitor intra-construct tetraloop–receptor association via FRET, and determined an upper limit of 12.7 kcal/mol in 10 mM MgCl2 (19). Using the aforementioned α-value, the average entropy contribution to forming the transition state (TΔS‡on) from the unbound starting state is small and slightly favorable: 2.4 (±0.5) kcal/mol. For dissociation (koff), the slopes are significantly steeper than the association plots, indicating even larger values for . The difference in association and dissociation transition state enthalpies is consistent with the binding exothermicity (Table 3). Also notable in Figure 5A is the difference between the [MgCl2]-dependence of the association and dissociation profiles. In plots of kon there exists a significant and systematic trend in the rate constants measured at a common temperature, manifested as a horizontal offset. However, for koff the temperature-dependent plots are nearly collinear. This behavior reflects a much more significant [MgCl2]-dependence in kon than in koff.Figure 5.


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)

Eyring plot of both association (kon) and dissociation (koff) rate constants measured in MgCl2 and KCl solutions. Each symbol represents the average rate constant measurement from a single titration, and error bars represent the measurement's true uncertainty as described in Supplementary Material. Circles, kon; squares, koff. For both rate constants, closed symbols reflect data that were obtained from titrations where Ka was measured directly (1 × 105 M−1 < Ka < 2 × 107 M−1), while for open symbols, Ka > 2 × 107 M−1. Lines were generated using best-fit regression parameters. (A) MgCl2 data, (B) KCl data. Y- and X-axis scaling are identical in the two panels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr894-F5: Eyring plot of both association (kon) and dissociation (koff) rate constants measured in MgCl2 and KCl solutions. Each symbol represents the average rate constant measurement from a single titration, and error bars represent the measurement's true uncertainty as described in Supplementary Material. Circles, kon; squares, koff. For both rate constants, closed symbols reflect data that were obtained from titrations where Ka was measured directly (1 × 105 M−1 < Ka < 2 × 107 M−1), while for open symbols, Ka > 2 × 107 M−1. Lines were generated using best-fit regression parameters. (A) MgCl2 data, (B) KCl data. Y- and X-axis scaling are identical in the two panels.
Mentions: Figure 5 plots the logarithms of kon and koff against inverse temperature for all of the experiments in this work. In MgCl2 (Figure 5A), the slopes (−ΔH‡/R) for each salt concentration and both reaction directions are negative, indicating that ΔH‡ is large and positive for both association and dissociation. The full set of transition state theory thermodynamic quantities are listed in Table 4. is on average 8.1 (±0.5) kcal/mol in MgCl2 solutions. This is consistent with a previous measurement, which used an oligonucleotide linker to monitor intra-construct tetraloop–receptor association via FRET, and determined an upper limit of 12.7 kcal/mol in 10 mM MgCl2 (19). Using the aforementioned α-value, the average entropy contribution to forming the transition state (TΔS‡on) from the unbound starting state is small and slightly favorable: 2.4 (±0.5) kcal/mol. For dissociation (koff), the slopes are significantly steeper than the association plots, indicating even larger values for . The difference in association and dissociation transition state enthalpies is consistent with the binding exothermicity (Table 3). Also notable in Figure 5A is the difference between the [MgCl2]-dependence of the association and dissociation profiles. In plots of kon there exists a significant and systematic trend in the rate constants measured at a common temperature, manifested as a horizontal offset. However, for koff the temperature-dependent plots are nearly collinear. This behavior reflects a much more significant [MgCl2]-dependence in kon than in koff.Figure 5.

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