""5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) is a hyperthermophilic and hyperthermostable enzyme belonging to purine nucleoside phosporylases, ubiquitous enzymes that function in the purine salvage pathway. SsMTAPII is a tightly packed hexameric protein organized as a dimer-of-trimers with one active site per monomer. Each subunit is stabilized by two pairs of intrasubunit disulfide bridges and contains in its C-terminal region a CXC motif as a typical feature. To get information on the role played by disulfide bonds in stability and folding, the conformational stability of SsMTAPII and C262S and C259S\\\/C261S mutants was studied by thermal and guanidinium chloride-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 reducing agents a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Differently 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 CXC disulfide on enzyme thermostability. Chemical and thermal unfolding of SsMTAPII are irreversible processes. Nevertheless, under reducing conditions we were able to highlight the reversible dissociation of the hexamer to monomers and to detect the reversibly unfolded species by SDS-PAGE. These results provide convincing evidence that disulfide bonds play a key role in the conformational stability of SsMTAPII. In fact, the removal of these covalent links weakening the highly compact structure of the enzyme allows the reversible transition from the native to the denatured state before covalent modifications, induced by high temperatures and chemical denaturants, may cause the irreversible protein degradation. This is the first report on the reversible unfolding of a hyperthermophilic oligomeric protein with disulfide bonds.""

THERMAL AND CHEMICAL UNFOLDING OF 5’-DEOXY-5’- METHYLTHIOADENOSINE PHOSPHORYLASE FROM THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS SOLFATARICUS

CACCIAPUOTI, Giovanna;PORCELLI, Marina
2012

Abstract

""5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) is a hyperthermophilic and hyperthermostable enzyme belonging to purine nucleoside phosporylases, ubiquitous enzymes that function in the purine salvage pathway. SsMTAPII is a tightly packed hexameric protein organized as a dimer-of-trimers with one active site per monomer. Each subunit is stabilized by two pairs of intrasubunit disulfide bridges and contains in its C-terminal region a CXC motif as a typical feature. To get information on the role played by disulfide bonds in stability and folding, the conformational stability of SsMTAPII and C262S and C259S\\\/C261S mutants was studied by thermal and guanidinium chloride-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 reducing agents a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Differently 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 CXC disulfide on enzyme thermostability. Chemical and thermal unfolding of SsMTAPII are irreversible processes. Nevertheless, under reducing conditions we were able to highlight the reversible dissociation of the hexamer to monomers and to detect the reversibly unfolded species by SDS-PAGE. These results provide convincing evidence that disulfide bonds play a key role in the conformational stability of SsMTAPII. In fact, the removal of these covalent links weakening the highly compact structure of the enzyme allows the reversible transition from the native to the denatured state before covalent modifications, induced by high temperatures and chemical denaturants, may cause the irreversible protein degradation. This is the first report on the reversible unfolding of a hyperthermophilic oligomeric protein with disulfide bonds.""
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11591/322070
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