title: Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.
abstract: 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 ΔHcal (850 kcal/mol) and Tmax (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.
authors.affiliation: Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Naples, Italy.
authors.forename: Maria Libera
authors.initials: ML
authors.lastname: Bagarolo
authors.affiliation: Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Naples, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
authors.affiliation: Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Naples, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Martino
authors.affiliation: Laboratory of Biochemistry, Centre for Protein Engineering, University of Liège, Liège B-4000, Belgium.
authors.forename: Georges
authors.initials: G
authors.lastname: Feller
authors.affiliation: Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Naples, Italy. Electronic address: giovanna.cacciapuoti@unina2.it.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000737:chemistry* / Q000201:enzymology; D013816:Thermodynamics; D013872:Thionucleosides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D013872:Thionucleosides; C021714:2-methylthioadenosine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D000241:Adenosine; D003545:Cysteine
keywords: 5′-Deoxy-5′-methylthioadenosine phosphorylase II from S. solfataricus; Differential scanning calorimetry; Disulfide bridges; Hyperthermophilic enzymes; Limited proteolysis; Protein thermostability