Formation of a Wrapped DNA–Protein Interface: Experimental Characterization and Analysis of the Large Contributions of Ions and Water to the Thermodynamics of Binding IHF to H′ DNA

Abstract To characterize driving forces and driven processes in formation of a large-interface, wrapped protein–DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H′ DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 °C. Both the binding constant K obs and enthalpy Δ H ° obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., Δ H o obs  = − 20.2 kcal·mol − 1 in 0.04 M KCl). Δ H ° obs increases linearly with [salt] with a slope (dΔ H ° obs /d[salt]), which is much larger in KCl (38 ± 3 kcal·mol − 1 M − 1 ) than in KF or KGlu (11 ± 2 kcal·mol − 1 M − 1 ). At 0.33 M [salt], K obs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK obs  = dln K obs /dln[salt] is almost twice as large in magnitude in KCl (− 8.8 ± 0.7) as in KF or KGlu (− 4.7 ± 0.6). A novel analysis of the large effects of anion identity on K obs , SK obs and on Δ H ° obs dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K obs , SK obs , and Δ H ° obs to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(± 0.2) × 10 3 ] from the 5340 A 2 of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F − and Glu − from this hydration water, relative to the situation with Cl − , which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K obs : dln K obs /d[GB]  = 2.7 ± 0.4 at constant KCl activity, indicating the net release of ca. 150 H 2 O molecules from anionic surface.