Unfolding and folding kinetics of amyotrophic lateral sclerosis-associated mutant Cu,Zn superoxide dismutases

More than 110 mutations in dimeric, Cu,Zn superoxide dismutase (SOD) have been linked to the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). In both human patients and mouse model studies, protein misfolding has been implicated in disease pathogenesis. A central step in understanding the misfolding/aggregation mechanism of this protein is the elucidation of the folding pathway of SOD. Here we report a systematic analyses of unfolding and folding kinetics using single- and double-jump experiments as well as measurements as a function of guanidium chloride, protein, and metal concentration for fully metallated (holo) pseudo wild-type and ALS-associated mutant (E100G, G93R, G93A, and metal binding mutants G85R and H46R) SODs. The kinetic mechanism for holo SODs involves native dimer, monomer intermediate, and unfolded monomer, with variable metal dissociation from the monomeric states depending on solution conditions. The effects of the ALS mutations on the kinetics of the holoproteins in guanidium chloride are markedly different from those observed previously for acid-induced unfolding and for the unmetallated (apo) forms of the proteins. The mutations decrease the stability of holo SOD mainly by increasing unfolding rates, which is particularly pronounced for the metal-binding mutants, and have relatively smaller effects on the observed folding kinetics. Mutations also seem to favour increased formation of a Zn-free monomer intermediate, which has been implicated in the formation of toxic aggregates. The results reveal the kinetic basis for the extremely high stability of wild-type holo SOD and the possible consequences of kinetic changes for disease.     

Equilibrium thermodynamic analysis of amyotrophic lateral sclerosis-associated mutant apo Cu,Zn superoxide dismutases

The folding and thermodynamic properties of metal free (apo) superoxide dismutases (SODs) are systematically analyzed using equilibrium guanidinium chloride (GdmCl) curves and differential scanning calorimetry (DSC). Chemically and structurally diverse amyotrophic lateral sclerosis (ALS)-associated mutations (G85R, G93R, E100G, I113T) are introduced into a pseudo-wild-type background that has no free cysteines, resulting in highly reversible unfolding. Analysis of the protein concentration dependence of GdmCl curves reveals formation of a monomer intermediate in equilibrium with native dimer and unfolded monomer. Global fitting of the data enables quantitative measurement of free energy changes for both dimer dissociation and monomer intermediate stability. All the mutations decrease protein stability, mainly by destabilizing the monomer intermediate, but also by tending to weaken dimerization, even for mutations far from the dimer interface. Thus, the effects of mutations seem to propagate through the apo protein, and result in increased population of both intermediate and unfolded monomers. This may underlie increased formation of toxic aggregates by mutants in ALS. Analysis of DSC data for apo SODs is consistent with stability measurements from GdmCl curves and provides further evidence for increased aggregation by mutant proteins through increased ratios of van't Hoff to calorimetric enthalpies of unfolding.     

Calorimetric analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-associated Gly-93 mutants of superoxide dismutase

Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i + 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N2) to two unfolded monomers (2U), N2 <--> 2U. The holo- and apo-forms of the mutants are significantly destabilized (by 1.5-3.5 kcal mol(-1) monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (DeltaHvH) to calorimetric (DeltaHcal) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, DeltaHvH is considerably larger than DeltaHcal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS.     

Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are > 95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼ 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

Destabilization of the dimer interface is a common consequence of diverse ALS‐associated mutations in metal free SOD1

Neurotoxic misfolding of Cu, Zn‐superoxide dismutase (SOD1) is implicated in causing amyotrophic lateral sclerosis, a devastating and incurable neurodegenerative disease. Disease‐linked mutations in SOD1 have been proposed to promote misfolding and aggregation by decreasing protein stability and increasing the proportion of less folded forms of the protein. Here we report direct measurement of the thermodynamic effects of chemically and structurally diverse mutations on the stability of the dimer interface for metal free (apo) SOD1 using isothermal titration calorimetry and size exclusion chromatography. Remarkably, all mutations studied, even ones distant from the dimer interface, decrease interface stability, and increase the population of monomeric SOD1. We interpret the thermodynamic data to mean that substantial structural perturbations accompany dimer dissociation, resulting in the formation of poorly packed and malleable dissociated monomers. These findings provide key information for understanding the mechanisms and energetics underlying normal maturation of SOD1, as well as toxic SOD1 misfolding pathways associated with disease. Furthermore, accurate prediction of protein–protein association remains very difficult, especially when large structural changes are involved in the process, and our findings provide a quantitative set of data for such cases, to improve modelling of protein association.     

Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability

Tetramerization of the human p53 tumor suppressor protein is required for its biological functions. However, cellular levels of p53 indicate that it exists predominantly in a monomeric state. Since the oligomerization of p53 involves the rate-limiting formation of a primary dimer intermediate, we engineered a covalently linked pair of human p53 tetramerization (p53tet) domains to generate a tandem dimer (p53tetTD) that minimizes the energetic requirements for forming the primary dimer. We demonstrate that p53tetTD self-assembles into an oligomeric structure equivalent to the wild-type p53tet tetramer and exhibits dramatically enhanced oligomeric stability. Specifically, the p53tetTD dimer exhibits an unfolding/dissociation equilibrium constant of 26 fM at 37 degrees C, or a million-fold increase in stability relative to the wild-type p53tet tetramer, and resists subunit exchange with monomeric p53tet. In addition, whereas the wild-type p53tet tetramer undergoes coupled (i.e. two-state) dissociation/unfolding to unfolded monomers, the p53tetTD dimer denatures via an intermediate that is detectable by differential scanning calorimetry but not CD spectroscopy, consistent with a folded p53tetTD monomer that is equivalent to the p53tet primary dimer. Given its oligomeric stability and resistance against hetero-oligomerization, dimerization of p53 constructs incorporating the tetramerization domain may yield functional constructs that may resist exchange with wild-type or mutant forms of p53.     

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.     

Quaternary structure of Dictyostelium discoideum nucleoside diphosphate kinase counteracts the tendency of monomers to form a molten globule

Multimeric enzymes that lose their quaternary structure often cease to be catalytically competent. In these cases, conformational stability depends on contacts between subunits, and minor mutations affecting the surface of the monomers may affect overall stability. This effect may be sensitive to pH, temperature, or solvent composition. We investigated the role of oligomeric structure in protein stability by heat and chemical denaturation of hexameric nucleoside diphosphate kinase from Dictyostelium discoideum and its P105G mutant over a wide range of pH. The wild-type enzyme has been reported to unfold without prior dissociation into monomers, whereas monomer unfolding follows dissociation for the P105G mutant (Giartosio et al. (1996) J. Biol. Chem. 271, 17845-51). We show here that these features are also preserved at alkaline pH, with the wild-type enzyme always hexameric at room temperature whereas the mutant dissociates into monomers at pH >or=10. In acidic conditions (pH 相似文献   

Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.     

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.     

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.     

Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Establishment of a cell model of ALS disease: Golgi apparatus disruption occurs independently from apoptosis

The Golgi apparatus (GA) appears disrupted in motor neurons of amyotrophic lateral sclerosis (ALS). Here, mouse motor neuron-like NSC-34 cell lines stably expressing human superoxide dismutase 1 (hSOD1)^wt and mutant hSOD1^G93A, as an ALS cell model, were constructed. The number of cells with disrupted GA increased from 14% to 34%. Furthermore, NSC-34/hSOD1^G93A cells showed lower levels of proliferation and differentiation. GA disruption was not caused by apoptosis as determined by several techniques including caspase-3 activation. Similarly, spinal cords from ALS patients did not show caspase-3 activation. Therefore, NSC-34/hSOD1^G93A cells are a suitable cell model to study GA dysfunction in ALS.     

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase

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.