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 temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.