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


Abstract

Domain II (residues 189-338, M(r) = 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 degrees 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 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(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 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 DeltaG(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. MARITIMA: domain II glutamate dehydrogenase. Study holds ProTherm entries: 7975, 7976, 7977 Extra Details: chimeric protein; domain exchange; molten globule state;,protein folding

Submission Details

ID: 36gdeTKE

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Consalvi V;Chiaraluce R;Giangiacomo L;Scandurra R;Christova P;Karshikoff A;Knapp S;Ladenstein R,Protein Eng. (2000) Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima. PMID:10906345
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Alpha-lactalbumin P00711 LALBA_BOVIN
99.3 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU
98.6 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
97.2 Alpha-lactalbumin P09462 LALBA_SHEEP
95.1 Alpha-lactalbumin P00712 LALBA_CAPHI
100.0 Alpha-lactalbumin P00709 LALBA_HUMAN
94.3 Alpha-lactalbumin P12065 LALBA_PAPCY
91.7 Alpha-lactalbumin P37154 LALBA_FELCA