title: Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase.
abstract: Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is -20.37(+/- 1.04) kcal (mol dimer)(-1). A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Anna-Karin E
authors.initials: AK
authors.lastname: Svensson
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel
authors.forename: Elena
authors.initials: E
authors.lastname: Kondrashkina
authors.forename: Jill A
authors.initials: JA
authors.lastname: Zitzewitz
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D001059:Apoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013482:Superoxide Dismutase / Q000737:chemistry*; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D001059:Apoproteins; D013482:Superoxide Dismutase