Protein Engineering, Vol. 13, No. 7, 501-507,
July 2000
© 2000 Oxford University Press
Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima
1 Dipartimento di Scienze Biochimiche `A. Rossi Fanelli', Università `La Sapienza', Piazzale A. Moro 5, 00185 Rome, Italy, 3 Institute of Organic Chemistry, Biophysical Chemistry Laboratory, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, 4 Department of Biosciences, NOVUM Karolinska Institutet, 14157 Huddinge, Sweden and 5 Department of Structural Chemistry, Discovery Research Oncology, Pharmacia and Upjohn, 20014 Nerviano (MI), Italy
Domain II (residues 189–338, Mr = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system 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 those observed in the crystal structure of the hexameric enzyme.The recombinant 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°C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70°C of 70.2 ± 4.0 kcal/mol and a 
Cp 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 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.maritima glutamate dehydrogenase is a thermostable protein with a Gmax 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.maritima domain II glutamate dehydrogenase.
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