Role of Water in Netropsin Binding to an A2T2 Hairpin DNA Site: Osmotic Stress Experiments

Abstract

The formation of two different minor groove complexes between netropsin and A2T2 DNA has been attributed to specific binding and hydration effects. In this study, we have examined the effect of added osmolyte (e.g., TEG or betaine) on the binding of netropsin to a hairpin DNA, d(CGCGAATTCGCGTC-TCCGCGAATTCGCG)-3, having a single A2T2 binding site. Netropsin binding to this DNA construct is described by a two fractional site model with a saturation stoichiometry of 1:1. Free energy changes, ΔGi, for formation of both complex I and complex II decrease continuously as osmolyte is added (e.g., ΔG1 decreases by 1.3 kcal/mol and ΔG2 decreases by 0.8 kcal/mol in 4 m osmolyte vs buffer). The negative ΔCp values for formation of both complexes, I and II, are largely unaffected by the addition of osmolyte. Formation of complex I is accompanied by the acquisition of 31 water molecules vs 19 waters for complex II. The most significant difference between the two osmolytes is that betaine diminishes the fractional formation of the complex II species, virtually eliminating complex II at 2 m. Addition of osmolyte or a decrease in the temperature have approximately the same effect on DNA hydration and on the thermodynamics of netropsin binding.