title: 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 (DeltaHobs) 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 degrees C, pH 8.1), and when possible, the binding free energy and entropy (DeltaG degrees obs, DeltaS degrees obs). At low monovalent salt concentrations (<0.1 M), the total DeltaHobs for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (DeltaHobs=-150(+/-5) kcal/mol). This is much larger than any DeltaHobs previously reported for a protein-nucleic acid interaction. However, at salt concentrations above 0.1 M, DeltaHobs is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (DeltaHobs=-70(+/-1)-kcal/mol in 2 M NaBr). These salt effects on DeltaHobs 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 DeltaHobs 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 DeltaHobs also are observed. Under conditions where we can determine DeltaG degrees obs, DeltaS degrees obs, and DeltaHobs (25 degrees 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 DeltaHobs (as well as DeltaG degrees obs and DeltaS degrees 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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Kozlov
authors.forename: T M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000838:Anions; D001426:Bacterial Proteins / Q000378:metabolism*; D002151:Calorimetry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D011485:Protein Binding; D012492:Salts / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D000838:Anions; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012492:Salts; D011071:Poly T; D012965:Sodium Chloride