Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M-r = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model sys tem to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E. coli using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to th ose observed in the crystal structure of the hexameric enzyme,The recombina nt domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH. range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C, Thermal unfolding of the protein was studied using dif ferential scanning calorimetry and circular dichroism spectroscopy. Both me thods yielded comparable values. The analysis revealed an unfolding enthalp y at 70 degrees C of 70.2 +/- 4.0 kcal/lmol and a Delta C-p value of 1.4 +/ - 0.3 kcal/mol K, Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kca/mol M in urea. The thermodynamic parameters for therm al and chemical unfolding equilibria indicate that domain II from T. mariti ma glutamate dehydrogenase is a thermostable protein with a Delta G(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T.mariti ma domain II glutamate dehydrogenase.