Calorimetric studies of E. coli SSB protein-single-stranded DNA interactions. Effects of monovalent salts on binding enthalpy

Abstract

Isothermal titration calorimetry (ITC) was used to examine the effects of monovalent salts (NaCl, NaBr, NaF and ChCl) on the binding enthalpy (ΔHobs) for E. coli SSB tetramer binding to the single-stranded oligodeoxythymidylates, dT(pT)69 and dT(pT)34 over a wide range of salt concentrations from 10 mM to 2.0 M (25°C, pH 8.1), and when possible, the binding free energy and entropy (ΔG°obs, ΔS°obs). At low monovalent salt concentrations (<0.1 M), the total ΔHobs for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (ΔHobs = −150(±5) kcal/mol). This is much larger than any ΔHobs previously reported for a protein-nucleic acid interaction. However, at salt concentrations above 0.1 M, ΔHobs is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (ΔHobs = −70(±1)-kcal/mol in 2 M NaBr). These salt effects on ΔHobs were mainly a function of anion type and concentration, with the largest effects observed in NaBr, and then NaCl, with little effect of [NaF]. These large effects of salt on ΔHobs appear to be coupled to a net release of weakly bound anions (Br− and Cl−) from the SSB protein upon DNA binding. However, at lower salt concentrations (<0.1 M), specific cation effects on ΔHobs also are observed. Under conditions where we can determine ΔG°obs, ΔS°obs, and ΔHobs(25°C, pH 8.1, 0.17 to 2 M NaBr), SSB binding to dT(pT)69 is enthalpically driven with a large unfavorable entropic contribution, both of which are dependent upon [NaBr]. These studies show that weak anion binding to a protein can result in large effects of salt concentration on ΔHobs (as well as ΔG°obs and ΔS°obs) for a protein-ssDNA interaction. The possibility of such effects needs to be considered in any interpretation of the thermodynamics of this and other protein-nucleic acid interactions.