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. 2012 Jan 10;51(1):565-72.
doi: 10.1021/bi201642p. Epub 2011 Dec 16.

Thermodynamics of ligand binding to a heterogeneous RNA population in the malachite green aptamer

Joshua E Sokoloski  1 , Sarah E Dombrowski, Philip C Bevilacqua
Affiliations

Thermodynamics of ligand binding to a heterogeneous RNA population in the malachite green aptamer

Joshua E Sokoloski et al. Biochemistry. .

Abstract

The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.

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Figures

Figure 1
Figure 1
Secondary and tertiary structures of the malachite green aptamer free and bound to MG and TMR. A.) Secondary structure of the aptamer. Key base interactions involved in the binding pocket are colored and correspond with panels C and D. B.) Structures of MG and TMR, which differ only in the presence of an ether linkage in TMR. C.) NMR solution structure of the MG-bound aptamer (1Q8N). MG is green spacefilling. D.) X-ray crystallography structure of the TMR-bound aptamer (1F1T). TMR is magneta spacefilling. Structures were rendered with PyMOL (Schrodinger, Cambridge MA).
Figure 2
Figure 2
ITC data for ligand binding to the MG aptamer. A.) 53.9 μM MG titrated into 4.36 μM MG aptamer at 25°C. B) 56.6 μM TMR titrated into 3.29 μM MG aptamer at 25°C. Note the greater dynamic range of heat for MG, which reflect its binding with much greater exothermicity. The x-axis is the molar ratio of ligand added to total RNA. Binding thermodynamic parameters are provided in Tables 1 and 2.
Figure 3
Figure 3
Temperature dependence of enthalpy for aptamer binding to MG and TMR. Circles represent average ΔH° values at a given temperature for MG (green) and TMR (magenta). Error bars are the standard error of the mean. Weighted linear regression of MG binding over the temperature range of 15 to 45 °C gives ΔH(T) = −1.13 kcal/mol°C (T − 25 °C) − 48.3 kcal/mol (R2 = 0.95), while that for TMR binding over the range 15 to 50 °C gives ΔH(T) = −0.45 kcal/mol°C (T − 25 °C) − 16.3 kcal/mol (R2 = 0.97). These plots provide ΔCp values for MG and TMR of −1.13+/−0.17 and −0.45+/−0.035 kcal K−1 mol−1, respectively. Note that the values of the final term in these fits are similar to the actual enthalpies for MG and TMR measured at 25 °C (provided in Tables 1 and 2), as expected. Buffer conditions were 10 mM sodium cacodylate (pH 5.8), 10 mM KCl, and 10 mM MgCl2.
Figure 4
Figure 4
Temperature dependence of entropy for aptamer binding to MG and TMR. Symbols represent average TΔS° values at each temperature for MG (green) and TMR (magenta). Error bars are from the standard error of the mean. Buffer conditions were 10 mM sodium cacodylate (pH 5.8), 10 mM KCl, and 10 mM MgCl2. Slope of the fits here is equal to ΔCp + ΔS, which is ~ΔCp because ΔS is relatively small. The slope of this plot is not interpreted in detail herein because ΔCp is directly obtainable in Figure 3 and ΔS is available in Tables 1 and 2. Nonetheless, this plot provides the trend of TΔS° with temperature.
Figure 5
Figure 5
Temperature dependence of apparent stoichiometry for aptamer binding to MG and TMR. Binding stoichiometries for the MG (green) and TMR (magenta) binding to MG aptamer were measured by ITC and are listed in Tables 1 and 2.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
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