THERMODYNAMIC CHARACTERIZATION OF NON-SEQUENCE-SPECIFIC DNA-BINDING BY THE SSO7D PROTEIN FROM SULFOLOBUS-SOLFATARICUS

We used isothermal titration calorimetry and fluorescence spectroscopy to investigate the thermodynamics of non-sequence-specific DNA-bindin g by the Sso7d protein from the archaeon Sulfolobus solfataricus. We r eport the Sso7d-poly(dGdC) binding thermodynamics as a function of buf fer composition (Tris-HCl or phosphate), temperature (15 to 45 degrees C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/(H2O)-H-2), and compare it to poly(dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represent ed by the McGhee-von Hippel model for non-cooperative binding, with a binding site size of four to five DNA base-pairs and binding free ener gies in the range Delta G degrees approximate to -7 to Delta G degrees approximate to -10 kcal mol(-1), depending on experimental conditions . The non-specific nature of the binding is reflected in similar therm odynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynam ics. Sso7d binding to poly(dGdC) is endothermic at 25 degrees C with a binding enthalpy Delta H degrees approximate to 10 kcal mol(-1) in bo th phosphate and Tris-HCl buffers at pH 7.6, indicating that Delta H d egrees does not include large contributions from coupled buffer ioniza tion equilibria at this pH. The binding enthalpy is temperature depend ent with a measured heat capacity change Delta C-o degrees = -0.25(+/- 0.01) kcal mol(-1) K-1 and extrapolations of thermodynamic data indica te that the complex is heat stable with exothermic binding close to th e growth temperature (75 to 80 degrees C) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on Delta G degr ees and the exchange of H2O for (H2O)-H-2 has only a small effect on D elta H degrees, consistent with the inference that complex formation i s not accompanied by net changes in surface hydration. Thus, other mec hanisms for the heat capacity change must be found. The observed therm odynamics is discussed in relation to the nature of non-sequence-speci fic DNA-binding by proteins. (C) 1998 Academic Press Limited.