Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase
| Autor: | Giovanna Cacciapuoti, Elisa Martino, Georges Feller, Maria Libera Bagarolo, Marina Porcelli |
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| Přispěvatelé: | Bagarolo, Maria Libera, Porcelli, Marina, Martino, Elisa, Feller, George, Cacciapuoti, Giovanna |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
5′-Deoxy-5′-methylthioadenosine phosphorylase II from S. solfataricu
Adenosine Hot Temperature Protein thermostability Protein Conformation medicine.medical_treatment ved/biology.organism_classification_rank.species Amino Acid Motifs Purine nucleoside phosphorylase Gene Expression Hyperthermophilic enzyme Biochemistry Analytical Chemistry Substrate Specificity Thermodynamic Enzyme Stability Disulfides Protein disulfide-isomerase Polyacrylamide gel electrophoresis Gel electrophoresis Thionucleosides Chemistry Sulfolobus solfataricus Recombinant Protein Recombinant Proteins Amino Acid Motif Thermodynamics Archaeal Proteins Molecular Sequence Data Biophysics Glycogen phosphorylase Disulfide Differential scanning calorimetry Archaeal Protein medicine Limited proteolysi Escherichia coli Disulfide bridge Cysteine Molecular Biology Kinetic Protease ved/biology Sulfolobus solfataricu Kinetics Purine-Nucleoside Phosphorylase Mutation Mutagenesis Site-Directed |
| Popis: | 5′-Deoxy-5′-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of Δ H cal (850 kcal/mol) and T max (39 °C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200–Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138–Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164. |
| Databáze: | OpenAIRE |
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