THERMAL UNFOLDING OF THE DNA-BINDING PROTEIN SSO7D FROM THE HYPERTHERMOPHILE SULFOLOBUS-SOLFATARICUS

Thermal unfolding of the small hyperthermophilic DNA-binding protein S so7d was studied by circular dichroism spectroscopy and differential s canning calorimetry. The unfolding transition can be described by a re versible two state process. Maximum stability was observed in the regi on between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperatu re between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and t he heat denatured states obtained by differential scanning calorimetry (620 cal/(mol K)) and circular dichroism spectroscopy (580 cal/(mol K )) resulted in comparable values. The thermodynamic reason for the hig h melting temperature of Sso7d is the shallow stability curve with a b road free energy maximum, corresponding to the relatively small heat c apacity change which was obtained. The calculated stability curve show s that Sso7d has, despite of its high melting temperature, an only mod erate intrinsic stability, which reaches its maximum (approximate to 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approxim ate to 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specifi c enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) va lues at convergence temperatures. No significant differences in therma l stability of the partially methylated Sso7d from Sulfolobus solfatar icus and the cloned non-methylated form of the protein expressed in Es cherichia coli were observed. (C) 1996 Academic Press Limited