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

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Abstract

Isothermal titration calorimetry (ITC) was used to examine the effects of monovalent salts (NaCl, NaBr, NaF and ChCl) on the binding enthalpy (ΔH(obs)) 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 ΔH(obs) for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (ΔH(obs) = -150 (±5) kcal/mol). This is much larger than any ΔH(obs) previously reported for a protein-nucleic acid interaction. However, at salt concentrations above 0.1 M, ΔH(obs) is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (ΔH(obs) = -70 (±1)-kcal/mol in 2 M NaBr). These salt effects on ΔH(obs) 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 ΔH(obs) 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 ΔH(obs) also are observed. Under conditions where we can determine ΔG°(obs) ΔS°(obs), and ΔH(obs) (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 ΔH(obs) (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.

Original languageEnglish
Pages (from-to)999-1014
Number of pages16
JournalJournal of Molecular Biology
Volume278
Issue number5
DOIs
StatePublished - May 22 1998

Keywords

  • Anion binding
  • Cooperativity
  • ITC
  • Oligomeric proteins
  • Thermodynamics

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