PMID:18237740
| Citation |
Vander Meulen, KA, Saecker, RM and Record, MT Jr (2008) 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. J. Mol. Biol. 377:9-27 |
|---|---|
| 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 degrees C. Both the binding constant K(obs) and enthalpy Delta H degrees (obs) depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., Delta H(o)(obs)=-20.2 kcal x mol(-1) in 0.04 M KCl). Delta H degrees (obs) increases linearly with [salt] with a slope (d Delta H degrees (obs)/d[salt]), which is much larger in KCl (38+/-3 kcal x mol(-1) M(-1)) than in KF or KGlu (11+/-2 kcal x 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)=dlnK(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 Delta H degrees (obs) dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K(obs), SK(obs), and Delta H degrees (obs) to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(+/-0.2)x10(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): dlnK(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. |
| Links |
PubMed PMC2336898 Online version:10.1016/j.jmb.2007.11.104 |
| Keywords |
Betaine/pharmacology; Calorimetry; DNA, Bacterial/chemistry; DNA, Bacterial/metabolism; Escherichia coli/drug effects; Escherichia coli/metabolism; Fluorescence Resonance Energy Transfer; Integration Host Factors/chemistry; Integration Host Factors/metabolism; Ions; Kinetics; Models, Molecular; Nucleic Acid Conformation/drug effects; Protein Binding/drug effects; Salts/pharmacology; Thermodynamics; Titrimetry; Water/metabolism |
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