Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus |
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Authors: | Giovanna Cacciapuoti Francesca Fuccio Luigi Petraccone Pompea Del Vecchio Marina Porcelli |
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Affiliation: | 1. Dipartimento di Biochimica e Biofisica “F. Cedrangolo”, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy;2. Consorzio Interuniversitario Biostrutture e Biosistemi “INBB”, Viale Medaglie d''Oro 305, Roma, Italy;3. Dipartimento di Chimica “Paolo Corradini”, Università di Napoli “Federico II”, Via Cintia, 80126 Napoli, Italy |
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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. |
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Keywords: | MTAP, 5&prime -deoxy-5&prime -methylthioadenosine phosphorylase SsMTAPII, 5&prime -deoxy-5&prime -methylthioadenosine phosphorylase II from Sulfolobus solfataricus PNP, purine nucleoside phosphorylase MTA, 5&prime -deoxy-5&prime -methylthioadenosine SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis CD, circular dichroism [θ], molar ellipticity per mean residue GdmCl, guanidinium chloride PIPES buffer, piperazine-N,N&prime -bis(ethanesulfonic acid) TCEP, Tris-(2-carboxyethyl) phosphine |
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