Kozlov A G, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA.
J Mol Biol. 1998 May 22;278(5):999-1014. doi: 10.1006/jmbi.1998.1738.
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.
等温滴定量热法（ITC）用于研究单价盐（NaCl、NaBr、NaF和氯化胆碱）在10 mM至2.0 M的宽盐浓度范围（25℃，pH 8.1）内对大肠杆菌单链DNA结合蛋白（SSB）四聚体与单链寡聚脱氧胸苷酸dT(pT)69和dT(pT)34结合焓（ΔHobs）的影响，并在可能的情况下研究结合自由能和熵（ΔG°obs、ΔS°obs）。在低单价盐浓度（<0.1 M）下，用单链DNA饱和SSB四聚体上所有位点的总ΔHobs对盐浓度的依赖性很小，但极大且放热（ΔHobs = -150(±5) kcal/mol）。这比先前报道的任何蛋白质 - 核酸相互作用的ΔHobs都要大得多。然而，在盐浓度高于0.1 M时，ΔHobs对NaCl和NaBr浓度相当敏感，随着盐浓度增加变得不那么负（在2 M NaBr中ΔHobs = -70(±1) kcal/mol）。这些盐对ΔHobs的影响主要是阴离子类型和浓度的函数，在NaBr中观察到的影响最大，其次是NaCl，[NaF]的影响很小。盐对ΔHobs的这些大影响似乎与DNA结合时SSB蛋白中弱结合阴离子（Br-和Cl-）的净释放有关。然而，在较低盐浓度（≤0.1 M）下，也观察到特定阳离子对ΔHobs的影响。在我们能够确定ΔG°obs、ΔS°obs和ΔHobs的条件下（25℃，pH 8.1，0.17至2 M NaBr），SSB与dT(pT)69的结合是由焓驱动的，伴有很大的不利熵贡献，两者都依赖于[NaBr]。这些研究表明，弱阴离子与蛋白质的结合可导致盐浓度对蛋白质 - 单链DNA相互作用的ΔHobs（以及ΔG°obs和ΔS°obs）产生很大影响。在解释这种及其他蛋白质 - 核酸相互作用的热力学时，需要考虑这种影响的可能性。
