title: Role of disulfide bonds in conformational stability and folding of 5'-deoxy-5'-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus.
abstract: Sulfolobus solfataricus 5'-deoxy-5'-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138-C205 and C200-C262) and a CXC motif (C259-C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.
authors: Dipartimento di Biochimica e Biofisica "F. Cedrangolo", Seconda Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy. giovanna.cacciapuoti@unina2.it
authors: Giovanna
authors: G
authors: Cacciapuoti
authors: Francesca
authors: F
authors: Fuccio
authors: Luigi
authors: L
authors: Petraccone
authors: Pompea
authors: P
authors: Del Vecchio
authors: Marina
authors: M
authors: Porcelli
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D004220:Disulfides; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase