combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 °C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb ~ 4.3 x 107 M-1, Hh ~ -7.5 kcal mol-1, ∆Sb ~ 9.3 cal K-1 mol-1, and ∆CP ~ 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 DSt.A3T2 complex yielded the following thermodynamic profile: Kb ~ 3.1 x 107 M-1, ∆Hb = -12.3 kcal mol-1, ∆Sb = -8 cal K-1 mol-1, and ∆Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of ~ 3.3 x 106 M-1, a more favorable ∆Hb of -18.8 kcal mol-1, a more unfavorable 8Sb of -34 cal K-1 mol-1, and a higher ∆Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 DSt.A3T2 complex.
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