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: 8366, 8367, 8368, 8369, 8370, 8371, 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379, 10530, 10531, 10532, 10533, 10534, 10535, 10536, 10537, 10538, 10539, 10540, 10541 Extra Details: additive : EDTA(100 uM), domain; glutamate dehydrogenase; hyperthermophiles; protein;,thermodynamic stability

Submission Details

ID: rvN2EXBa

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 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

Study Summary

Number of data points 60
Proteins Glutamate dehydrogenase ; Glutamate dehydrogenase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Tm buffers:phosphate: 20 mM, pH:8.0, prot_conc:- ; Experimental Assay: dHvH buffers:phosphate: 20 mM, pH:8.0, prot_conc:- ; Experimental Assay: Tm pH:7.55, buffers:phosphate: 20 mM, prot_conc:- ; Experimental Assay: dHvH pH:7.55, buffers:phosphate: 20 mM, prot_conc:- ; Experimental Assay: Tm buffers:phosphate: 20 mM, pH:7.15, prot_conc:- ; Experimental Assay: dHvH buffers:phosphate: 20 mM, pH:7.15, prot_conc:- ; Experimental Assay: Tm buffers:phosphate: 20 mM, pH:6.6, prot_conc:- ; Experimental Assay: dHvH buffers:phosphate: 20 mM, pH:6.6, prot_conc:- ; Experimental Assay: Tm pH:6.3, buffers:phosphate: 20 mM, prot_conc:- ; Experimental Assay: dHvH pH:6.3, buffers:phosphate: 20 mM, prot_conc:- ; Experimental Assay: Tm buffers:phosphate: 20 mM, pH:5.9, prot_conc:- ; Experimental Assay: dHvH buffers:phosphate: 20 mM, pH:5.9, prot_conc:- ; Experimental Assay: dHcal prot_conc:0.019 mM, buffers:phosphate: 20 mM, pH:8.0 ; Experimental Assay: Tm prot_conc:0.019 mM, buffers:phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHvH prot_conc:0.019 mM, buffers:phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHcal pH:7.5, buffers:phosphate: 20 mM, prot_conc:0.059 mM ; Experimental Assay: Tm pH:7.5, buffers:phosphate: 20 mM, prot_conc:0.059 mM ; Experimental Assay: dHvH pH:7.5, buffers:phosphate: 20 mM, prot_conc:0.059 mM ; Experimental Assay: dHcal prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHvH prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHcal pH:6.5, buffers:phosphate: 20 mM, prot_conc:0.060 mM ; Experimental Assay: Tm pH:6.5, buffers:phosphate: 20 mM, prot_conc:0.060 mM ; Experimental Assay: dHvH pH:6.5, buffers:phosphate: 20 mM, prot_conc:0.060 mM ; Experimental Assay: dHcal prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:6.5 ; Experimental Assay: Tm prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:6.5 ; Experimental Assay: dHvH prot_conc:0.033 mM, buffers:phosphate: 20 mM, pH:6.5 ; Experimental Assay: dHcal pH:6.0, prot_conc:0.029 mM, buffers:phosphate: 20 mM ; Experimental Assay: Tm pH:6.0, prot_conc:0.029 mM, buffers:phosphate: 20 mM ; Experimental Assay: dHvH pH:6.0, prot_conc:0.029 mM, buffers:phosphate: 20 mM ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: Tm pH:7.55, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:7.15 ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:6.6 ; Experimental Assay: Tm prot_conc:0.2 mg/ml, pH:6.3, buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:5.9 ; Experimental Assay: dHcal prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHvH prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHcal pH:7.5, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm pH:7.5, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHvH pH:7.5, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHcal prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHvH prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHcal prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:6.5 ; Experimental Assay: Tm prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:6.5 ; Experimental Assay: dHvH prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM, pH:6.5 ; Experimental Assay: dHcal pH:6.0, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm pH:6.0, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHvH pH:6.0, prot_conc:0.2 mg/ml, buffers:Sodium phosphate: 20 mM ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O
Libraries Mutations for sequence MPEKSLYEMAVEQFNRAASLMDLESDLAEVLRRPKRVLIVEFPVRMDDGHVEVFTGYRVQHNVARGPAKGGIRYHPDVTLDEVKALAFWMTWKTAVMNLPFGGGKGGVRVDPKKLSRNELERLSRRFFSEIQVIIGPYNDIPAPDVNTNADVIAWYMDTYSMNVGHTVLGIVTGKPVELGGSKGREEATGRGVKVCAGLAMDVLGIDPKKATVAVQGFGNVGQFAALLISQELGSKVVAVSDSRGGIYNPEGFDVEELIRYKKEHGTVVTYPKGERITNEELLELDVDILVPAALEGAIHAGNAERIKAKAVVEGANGPTTPEADEILSRRGILVVPDILANAGGVTVSYFEWVQDLQSFFWDLDQVRNALEKMMKGAFNDVMKVKEKYNVDMRTAAYILAIDRVAYATKKRGIYP

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 Glutamate dehydrogenase P96110 DHE3_THEMA