title: Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s).
abstract: Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis.
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30043, Taiwan.
authors.forename: Hui-Chu
authors.initials: HC
authors.lastname: Hsieh
authors.forename: Thallapuranam Krishnaswamy S
authors.initials: TK
authors.lastname: Kumar
authors.forename: Chi-Cheng
authors.initials: CC
authors.lastname: Chiu
authors.forename: Chin
authors.initials: C
authors.lastname: Yu
mesh_terms: D019281:Dimerization; D004789:Enzyme Activation; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000032:analysis / Q000138:chemical synthesis*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013482:Superoxide Dismutase / Q000032:analysis / Q000737:chemistry*; D044149:Vibrio alginolyticus / Q000201:enzymology*
chemical_list: D046912:Multiprotein Complexes; D013482:Superoxide Dismutase; D019791:Guanidine