Binding-linked protonation of a DNA minor-groove agent

Abstract

The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK(a) of 6.3 that is susceptible to a protonation process. Spectroscopic studies indicate an increase of 2.7 pK(a) units in the linking group when the compound binds to an A/T minor-groove site. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. The two methods established a proton-uptake profile as a function of pH. The exothermic enthalpy of complex formation varies with different pH conditions. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Energetic contributions from different factors were also estimated for the ligand/DNA complex.

FIGURE 1

Structure of CGP 40215A.

FIGURE 2

Fractional absorbance change of the free (▪) and bound (•) ligand, and the fractional proton uptake (○). Solid lines are the fits using Eq. 1; the pK_a values are 6.3 ± 0.1 and 9.0 ± 0.1 for the free and bound ligands, respectively. Maximum proton uptake occurs at pH 7.7. Calorimetric titration results at two pH values (□) are also plotted, illustrating a good agreement.

FIGURE 3

ITC titrations of CGP with poly(dAdT) · poly(dAdT) in MES and cacodylate buffers. Top panel is a representative titration curve of 3-μL injections of 0.473 mM CGP into 0.12 mM DNA base pairs in cacodylate buffer at pH 6.25, 25°C. Bottom panel is a plot of integrated heats per mol of CGP in cacodylate buffer (•) and in MES buffer (▪) versus the ratio of the ligand to DNA base pairs. The primary binding occurs at a ratio of 0.125 that is equivalent to one compound per eight base pairs. It can be seen that 1), a significant difference in the heat release occurs at the primary binding between the two buffers under the same conditions; and 2), secondary binding to the polymer occurs at higher ratios in both buffers.

FIGURE 4

An isothermal calorimetric titration curve of CGP 40215A into AATT hairpin solution. Every peak represents the heat released from a 5-μl injection of 50 μM CGP into 2 μM oligomer concentration in MES buffer with 0.1 M NaCl, pH 6.25 at 298 K. The corrected heat was fitted to obtain thermodynamic binding parameters.

FIGURE 5

Effects of solution pH on the heat capacity change. In MES buffer (10 mM MES, 0.1 M NaCl, 1 mM EDTA, pH 6.25), linear fitting of the observed binding enthalpy as a function of temperature yields a slope of –155 ± 16 cal/(mol·K). The slope becomes more negative (−234 ± 11 cal/(mol·K)) in acetate buffer (10 mM acetate, 0.1 M NaCl, 1 mM EDTA, pH 5.00), where proton linkage is minimized.

FIGURE 6

SPR binding affinities. Top panel is a plot of sensorgrams for the interaction of CGP with 5′biotin-CGAATTCGTCTCCGAATTCG-3′ hairpins in HEPES buffer at pH 7.45, 0.2 M NaCl, and 25°C. Bottom panel is a plot of stoichiometry-normalized binding isotherms at three different NaCl concentrations: 0.1 M, 0.2 M, and 0.4 M.

FIGURE 7

Binding affinities at different salt concentrations and two different pHs. The dependence of free energies on salt concentrations at pH 6.25 and pH 7.45. The slopes are −2.1 at pH 6.25 and −2.0 at pH 7.45. Using these slopes and the ligand charges, the counterion density per phosphate at the binding site is 0.68 ± 0.02.

FIGURE 8

Solvent-accessible surface area of the complex generated by the GRASP program. A view is at the minor groove with the ligand bound at the AATT site. The polar atoms are in red and nonpolar atoms are in white. Arrays of polar phosphate-bound oxygen atoms are visible along the DNA backbone. The red areas in the minor groove are the nitrogen, and nitrogen-bound hydrogen atoms of the ligand. The accessible surface was generated using Cornell et al radii (39) with a probe radius of 1.7683 Å.