title: Refolding pathway and association intermediates of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.
abstract: The denaturation and renaturation processes of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been investigated using guanidinium chloride as denaturant. The enzyme is highly stable and the transition midpoint for guanidinium chloride denaturation is 6.1 M. The recovery of enzyme structure occurs after dilution of the denaturant at 20 degrees C through the formation of structured monomers. Concentration of the structured monomers leads to the formation of higher association states with a tertiary structure different from that of the native enzyme. Activity is observed only in the presence of the hexamers, although a heating step at 70 degrees C is required to fully reactivate the hexamer formed at 20 degrees C. The refolding process and the intermediate(s) were studied by activity assay, spectroscopic methods, size-exclusion chromatography, and ultracentrifugation analysis.
authors.affiliation: Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Consalvi
authors.forename: R
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: S
authors.initials: S
authors.lastname: Millevoi
authors.forename: A
authors.initials: A
authors.lastname: Pasquo
authors.forename: P
authors.initials: P
authors.lastname: Vecchini
authors.forename: E
authors.initials: E
authors.lastname: Chiancone
authors.forename: R
authors.initials: R
authors.lastname: Scandurra
mesh_terms: D001105:Archaea / Q000201:enzymology*; D002942:Circular Dichroism; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D006146:Guanidines; D005969:Glutamate Dehydrogenase; D019791:Guanidine