A HIGHLY SALT-DEPENDENT ENTHALPY CHANGE FOR ESCHERICHIA-COLI SSB PROTEIN-NUCLEIC ACID-BINDING DUE TO ION-PROTEIN INTERACTIONS

We have examined the linkage between salt concentration and temperatur e for the equilibrium binding of the tetrameric Escherichia coli singl e-stranded binding (SSB) protein to three single-stranded nucleic acid s, poly(U), dA(pA)(69), and dT(pT)(69), by van't Hoff analysis and iso thermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)(65) mode, the equilibrium association constant, K-obs, decrease s with increasing salt concentration-at all temperatures examined, and binding is enthalpy-driven; however, the value of partial derivative log K-obs/partial derivative log [NaCl] is highly temperature-dependen t, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 deg rees C. This indicates that Delta H-obs for SSB-poly(U) binding is str ongly dependent on [NaCl]; based on van't Hoff analyses, Delta H-obs v aries from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 0.42 M NaCl (partial derivative Delta H-obs/partial derivative log [Na Cl] = 60 +/- 5 kcal/mol). However, partial derivative Delta H-obs/part ial derivative log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on Delta H-obs is due primari ly to Cl-. Similar effects were also observed for SSB binding to dA(pA )(69). We also measured Delta H-obs and its dependence on [NaCl] for S SB binding to dT(pT)(69) by ITC and find Delta H-obs = -144 +/- 4 kcal /mol (0.175 M NaCl, pH 8.1, 25 degrees C) and partial derivative Delta H-obs/partial derivative log [NaCl] = 46 +/- 2 kcal/mol (0.175 - 2.0 M NaCl). effects of [NaCl] on Delta H-obs appear to result, at least p artly, from the release of preferentially bound Cl- from SSB protein u pon binding nucleic acid, with the release of Cl- being linked to a pr ocess with Delta H > > 0. Effects of salt concentration on Delta H-obs are not observed for processes in which only monovalent cations are r eleased from the nucleic acid, presumably since Na+ or K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such sal t effects on Delta H-obs may serve as a signature for differential ion -protein binding. These results underscore the need to examine the lin kage of [salt] to Delta H-obs, as well as Delta G degrees(obs) and Del ta S degrees(obs), in order to understand the bases for stability and specificity of protein-nucleic acid interactions.