The role of the salt concentration, proton, and phosphate binding on the thermal stability of wild and cloned DNA-binding protein Sso7d from Sulfolobus solfataricus

Abstract

The acidic pH (1.5–7.0) and ionic strength (0.005–0.2 M) dependence of thermodynamic functions of protein Sso7d from Sulfolobus solfataricus, cloned (c-Sso7d) and N-heptapeptide deleted [c-des(1–7)Sso7d] in glycine, and phosphate buffers was studied by means of adiabatic scanning calorimetry. The difference of proton binding was estimated from Δ H cal(pH), T d(pH), and ( δT d/ δpH). It was found that a single group non-co-operative ionization with apparent p K a=3.25 for both cloned and deleted proteins govern the thermal unfolding of two different (protonated and unprotonated) forms. Δ H° is found to be pH-independent and the changes in stability (Δ G°) originate from changes in entropy terms. The apparent p K a measured at high salt concentrations decreases with 0.5 pH units from glycine to phosphate and the free energy of transfer at high ionic strength is 0.7 kcal/mol. The ionic strength dependence for the pH-dependent D-states is very different at pH 6.0 and 1.5. This is consistent with the property of denatured state to be more compacted or “closed” ( D c) at neutral or weak acidic pH and more random or “open” ( D o) at acidic pH. From the Bjerrum’s relation was found the number of screened charges important for the unfolding process. The main conclusions are: (1) the thermal stability of Sso7d has prominently entropic nature; (2) a single non-co-operative ionization controls the conformations in the D-state; and (3) pH-dependent conformational equilibrium could be functionally important in Sso7d-DNA recognition.