Sso10a, a hyperthermophile DNA-binding protein

Hyperthermophile proteins have important attributes that can be utilized to enhance the potential for rational manipulation of protein structure and stability. Sso10a is one of the small highly basic DNA binding proteins (7, 8 and 10 kDa) from Sulfolobus solfataricus that were initially thought to be involved in DNA packaging. A number of structural and thermodynamic studies have been conducted on the 7 kDa protein. Sso10b and Sso10b2 were also characterized to a reasonable extent. This work addresses the structure of Sso10a and the contribution of important electrostatic interactions in the stability of the protein. The gene for the protein was cloned and expressed and the protein was purified using cation exchange chromatography. The protein was shown to be a dimer at physiological pH, but exists mainly as a monomer below pH 3 (1mM glycine, 20°C). The thermal and chemical denaturation-induced unfolding of Sso10a was found to be essentially completely reversible under wide pH and salt conditions. The solution NMR structure of the protein was obtained and consists of two winged-helix domains separated by a two-stranded antiparallel coiled coil rod. The crystal structure was obtained in collaboration with Dr. Liqing Chen (University of Alabama in Huntsville) and the protein was found to have similar structure in solution and in crystal. An inter-monomer H-bonded salt bridge is seen at an unusual position in the coiled coil region of the structure. D69A, K86A single mutants and D69A-K86A double mutants were constructed to study the importance of the salt bridge in the stability of the protein. The structure of the protein was found unaltered by the mutations. The stability curves of Sso10a and the mutants were defined in high salt conditions using chemical denaturation and in low salt conditions using differential scanning calorimetry. Under high salt conditions, the salt-bridge was shown not to be important in the formation or stability of the protein. Under low salt conditions, the stability of the double mutant was significantly lower and the change cannot be accounted for by the disruption of a single salt bridge. The importance of the salt bridge in the stability of the protein was higher in low ionic strength conditions than in high ionic strength solution.