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.
The acidic pH (1.5-7.0) and ionic strength (0.005-0.2M) 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 deltaHcal(pH), Td(pH), and (deltaTd/deltapH). It was found that a single group non-co-operative ionization with apparent pKa = 3.25 for both cloned and deleted proteins govern the thermal unfolding of two different (protonated and unprotonated) forms. deltaH degrees is found to be pH-independent and the changes in stability (deltaG degrees ) originate from changes in entropy terms. The apparent pKa 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" (Dc) at neutral or weak acidic pH and more random or "open" (Do) 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.
Todorova
Roumiana
R
Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. todorova@obzor.bio21.bas.bg
Atanasov
Boris
B
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005998:Glycine; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism*; D011522:Protons; D012492:Salts / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000737:chemistry*
D019843:Archaeal Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; D010710:Phosphates; D011522:Protons; D012492:Salts; C093228:Sso7d protein, Sulfolobus; D005998:Glycine