Metal Content of Protein Preparations
The metal contents of the purified SOD1 proteins that were metalated in vivo
and used in this study as they were isolated (“as isolated” proteins) are presented in . In addition to the wild-type protein, they include proteins with mutations related to ALS and “AS” proteins lacking free cysteines. The AS mutant, C6A/C111S, is a “pseudo-WT” SOD1 in which both free cysteines have been removed by mutation, with the buried cys6 mutated to alanine and the surface cys111 changed to serine. AS/A4V, AS/G93A and AS/G85R have ALS-related mutations in the same AS background. These AS proteins have been used by many investigators studying SOD1; AS-SOD1 has a stability similar to that of WT SOD1 but melts reversibly, while WT-SOD1 melts irreversibly, presumably because of disulfide-induced aggregation following thermal unfolding 
. None of the proteins analyzed in this study had a full complement of copper and zinc, consistent with previous reports using similarly expressed and purified SOD1 
). The zinc contents of most of the proteins were close to or greater than two per dimer; SOD1 is frequently isolated with zinc binding partially to the copper sites of the enzyme 
in addition to the normal zinc binding sites. The copper content was lower, ranging from 0.07 to 0.74 per dimer ().
Metal contents of SOD1 preparations.
As stated in the Introduction, the beta barrel and dimer interface mutants belong to the class of “wild-type-like” mutants, which have metal content and catalytic activity close to that of WT SOD1, while “metal-binding-region” mutants have significantly reduced metal content and catalytic activity. In agreement with previous results, all the beta barrel mutants except L38V had a total of 2.8 to 3.0 metals per dimer, close to the value of 3.54 for WT. Of the metal-binding-region mutants D125H, H46R and H80R had greatly reduced metal contents (with only 25% of the metal sites occupied in H80R), while S134N and G85R had metal contents close to that of WT. Of the two dimer interface mutants, A4V was similar in metal content to other wild-type-like mutants, while I149T had a lower metal content. Three of the mutants lacking free sulfhydryl groups, AS, AS/A4V and AS/G93A, had total metal contents similar to the corresponding proteins with a normal cysteine content, while the metal content of AS/G85R was lower than that of G85R.
We prepared metal-free apo-proteins by extended dialysis against EDTA. ICP-MS analysis on individual preparations confirmed that extensive dialysis against EDTA effectively and reproducibly removed all the Cu and Zn from the enzyme.
In order to obtain a set of ALS mutant and WT SOD1 proteins with uniform contents of Cu and Zn, we attempted to remetalate the apo-SOD1 preparations by a published method that has been frequently used successfully with SOD1 expressed in bacteria 
. We found, however, that this procedure failed to produce either fully metalated or fully active SOD1 proteins. We found alternative remetalation procedures that led successfully to fully metalated and fully active proteins, but these preparations failed to form any aggregates under conditions that promoted aggregate formation from the as-isolated preparations (data not shown). The failure to aggregate could be attributed to irreversible oxidation of one of the free cysteine residues. (See Text S1
and Figure S1
for details.) We therefore concluded that the use of remetalated proteins for in vitro
aggregation studies was not feasible. For most experiments in this study, we instead used the “as-isolated” proteins which had been metalated in yeast prior to isolation. These preparations will be referred to as “metalated” SOD1 in the discussion that follows.
Metalated WT SOD1 Forms Amyloid at pH 3
SOD1 solutions were incubated in the presence of 10 µM ThT, a dye that binds specifically to amyloid structures 
. Incubations were performed in a microplate reader, as described in “Materials and Methods
”, and formation of amyloid was monitored by the increase in ThT fluorescence. When WT SOD1 at a concentration of 1 mg/ml (32 µM dimer or 65 ) was incubated at either 24°C or 37°C in 50 mM sodium citrate, 0.1 M NaCl, 1 M guanidine hydrochloride, pH 3, the fluorescence remained close to zero for a lag time of a few hours, then increased until a plateau was reached. (). The fluorescence values at the plateau were similar to those observed in incubations of α-synuclein at the same protein concentration under conditions which have been shown to promote formation of amyloid 
. Similar results were obtained when the buffer was formate instead of citrate, a finding which rules out the possibility that amyloid formation at pH 3 requires the presence of a buffer anion capable of coordinating copper or zinc. The amyloid character of the incubation product was confirmed by a variety of morphological and spectroscopic criteria (see below). In the remainder of this article, we use the term “amyloid” to refer to these aggregates. In incubations for as long as one week, amyloid formation was observed only when the solutions were agitated with a Teflon ball. The plateau fluorescence was higher and the lag time shorter at 37°C than at 24°C. It should be noted that amyloid formation from SOD1 at acidic pH (3.5) was also reported by DiDonato et al. 
Effect of incubation conditions on amyloid formation.
At both temperatures amyloid was formed at guanidine concentrations ranging from 0.5 to 2 M, but not at 3 M (data for 37°C is shown in ). Since the lag time was longer at 0.5 M guanidine, a concentration of 1 M and a temperature of 37°C were used in most experiments. No amyloid formed when NaCl was substituted for guanidine hydrochloride (data not shown), indicating that the effect of guanidine hydrochloride is related to its chaotropic properties and not simply to its effect on the ionic strength.
Amyloid formed within a restricted pH range. Much less amyloid formed when the pH was increased from 3 to 4, and only a very small amount of amyloid formed at pH 5 with a much longer lag time (). No amyloid formed at pH 2 or at pH 6 and above.
Acetonitrile could substitute for guanidine hydrochloride in promoting amyloid formation. Concentrations of 10–30% acetonitrile (but not higher concentrations) were effective (). Trifluoroethanol at concentrations from 10 to 30% did not promote amyloid formation (data not shown).
Removal of Copper and Zinc Allows Amyloid Formation at Neutral pH
Because acidic pH causes the dissociation of metals from SOD1, we reasoned that the ability of WT SOD1 to form amyloid at low pH could be due, at least in part, to the loss of metal ions. We hypothesized that metal-free SOD1 might therefore be able to form amyloid at higher values of pH that are more physiologically relevant. The kinetic parameters for amyloid formation from metalated and apo-SOD1 at various pH values in the presence of 1 M guanidine are summarized in . As shown above (), the yield of amyloid from metalated WT-SOD1 was sharply dependent on pH, with considerably less amyloid at pH 4 or 5 compared to pH 3 and no amyloid formed at pH 6 or 7. In contrast, there was little variation in either the amplitude or the lag time for apo-SOD1 as the pH was varied between 3 and 7. This suggests that acidic pH is needed for amyloid formation mainly because it promotes the loss of metals. Most strikingly, apo-SOD1 formed amyloid at pH 6 and 7, while no conditions have been found which allow amyloid formation from metalated SOD1 with its intramolecular disulfide bond intact at pH greater than 5.
Kinetic parameters of amyloid formation from metalated and apo-SOD1 in 1 M guanidine.
Apo-SOD1 also formed amyloid at pH 7 in the absence of guanidine. The lag times were longer than those observed for apo-SOD1 with 1 M guanidine, and the increase in fluorescence was slower and more gradual (). For most samples, the ThT fluorescence was still increasing after 300 hours. Addition of acetonitrile had no effect on the time course of amyloid formation from apo-SOD1 at pH 7 (data not shown).
Amyloid formation from apo- and reduced WT SOD1 at pH 7.
Amyloid formed from apo-SOD1 at pH 7 was stabilized against disassembly by intermolecular disulfide bonds. When the aggregated apo-WT-SOD1 was recovered by pelleting and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein entered the gel only when mercaptoethanol was present in the sample buffer (, lanes 6 and 7). This is in contrast to amyloid formed at pH 3 and 1 M guanidine, in which the monomer band was only slightly less intense under nonreducing conditions than under reducing conditions, demonstrating that little if any intermolecular disulfides had formed at pH 3 (, lanes 4 and 5). This pH dependence is in accord with the well-characterized pH dependence of disulfide formation 
Detection of intermolecular disulfide bonds in SOD1 amyloid prepared under different conditions.
Reduction of the Intramolecular Disulfide Bond Promotes Amyloid Formation from Metalated SOD1
The intramolecular disulfide bond of SOD1 can be cleaved by extended incubation with high concentrations of reducing agents 
, converting SOD1S-S
. When SOD1 was incubated with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and 1 M guanidine at neutral pH, there was a lag of about 45 hours followed by a rise in ThT fluorescence (); no amyloid was formed at pH 7 with 10 mM TCEP or 1 M guanidine alone. The use of 0.1 M TCEP allowed slow formation of amyloid at pH 7 in the absence of guanidine.
The presence of disulfide bonds in the aggregated material was dependent on the TCEP concentration during the incubation. Amyloid prepared in 1 M guanidine and 10 mM TCEP showed very little material entering an SDS gel unless mercaptoethanol was present (, lanes 8 and 9). On the other hand, when the TCEP concentration was 0.1 M, protein from the aggregates readily entered the gel (, lanes 2 and 3), indicating that SOD1 can form amyloid lacking disulfide bonds at neutral pH. Apparently, at the lower concentration TCEP initially cleaves the intramolecular disulfide bond but then is consumed as a result of reaction with dissolved oxygen, after which intermolecular disulfide bonds are allowed to form, while at the higher TCEP concentration the persistence of reduced TCEP throughout the incubation insures that only amyloid lacking intermolecular disulfide bonds is produced. The ability of SOD1 to form amyloid lacking intermolecular disulfide bonds is also suggested by the fact that AS-SOD1, which lacks two of the four cysteines, forms amyloid in greater yield than WT SOD1 upon incubation with 0.1 M TCEP (see “Effect of Mutations on Fibrillation of Metalated SOD1 in 0.1 M TCEP at pH 7” below).
Electron Microscopy and Atomic Force Microscopy of Amyloid
The amyloid character of the ThT-positive aggregates was confirmed by electron microscopy. After incubation in 1 M guanidine at pH 3 and 37°C, nearly all of the protein was present as fibrils with diameters of 5 to 10 nm (some as large as 14 nm) and lengths typically 0.5 to 3 µm (). Often the fibrils aggregated with each other to form a meshlike network (). In some samples short fibrils having a distinct substructure were observed (). The morphology of fibrils prepared from WT SOD1 and from ALS mutant SOD1 proteins were similar. Atomic force microscopy of these preparations revealed fibrillar structures with diameters ranging from 4–14 nm (average 7.9±2.6) and lengths ranging from 0.5–3 µm (Figure S2
). There appeared to be a bimodal distribution of diameters, with fibrils of 5 nm or 10 nm in diameter being most common.
Electron microscopy of SOD1 amyloid formed at pH 3.
Under conditions which were less than optimal for amyloid formation, as measured by the plateau value of the ThT fluorescence, nonfibrillar material was also observed in electron micrographs. For example, when the incubation was performed at 24°C at pH 3, we frequently observed spherical structures with an average diameter of about 13 nm (), often together with fibrils () or weblike networks of thinner, less well-defined fibrillar material ().
Amyloid fibrils were also observed in aggregates derived from apo-SOD1 and disulfide-reduced SOD1. Long fibrils, short fibrils and meshlike networks were observed under different incubation conditions (Figure S3
). Spherical structures were present in amyloid prepared from apo-SOD1 but not in amyloid prepared from TCEP-treated SOD1.
Spectroscopic Characterization of Amyloid
The amyloid character of the aggregated material was verified by a number of spectroscopic methods. Binding of the amyloid-specific dye Congo red was demonstrated both by a shift in the absorbance maximum of the dye 
) and by birefringence as monitored by polarization microscopy 
). In addition, circular dichroism (CD) (Figure S4C
) and infrared (IR) (Figure S4D
) spectra characteristic of amyloid were observed. The trough in the circular dichroism spectrum shifted from 209 nm for soluble SOD1 to 220–222 nm for SOD1 amyloid. A similar spectrum has been observed for amyloid formed from a number of other proteins 
. The infrared amide I peak shifted from 1644 cm−1
to 1631 cm−1
, indicating a significant reorganization of the beta sheet characteristic of amyloid 
. Similar spectra were obtained for amyloid prepared from both metalated and apo-SOD1 under a variety of incubation conditions. This suggests that amyloid produced under different sets of conditions have a common structural organization. Fluorescence data indicate that tryptophan 32 becomes buried upon amyloid formation and that SOD1 amyloid does not have significant amounts of solvent-exposed hydrophobic clusters (see Text S2
ALS Mutations Enhance Amyloid Formation Under Some but Not All Incubation Conditions
In order to examine the effect of ALS mutations on the tendency of SOD1 to form amyloid, we compared the properties of wild-type and mutant proteins under each of the three sets of conditions found to promote amyloid formation from WT SOD1.
Effect of Mutations on Fibrillation of Metalated SOD1 at Acidic pH
ALS mutations did not stimulate amyloid formation from metalated SOD1 in 1 M guanidine at pH 3. On the contrary, the yield of amyloid was as great for WT SOD1 as for any of the mutants tested, and the lag time for WT SOD1 was among the shortest of any of the proteins examined (see ).
Kinetic Parameters for Amyloid Formation from Metalated SOD1 at Acidic pH
At pH 5, however, many ALS mutants showed enhanced amyloid formation compared to WT SOD1. Representative fluorescence time courses for selected mutants are shown in , and a complete set of kinetic parameters is presented in . WT SOD1 forms barely detectable amounts of amyloid at this pH (). In 1 M guanidine, four out of five metal-binding-region mutants – D125H, G85R, H46R, and H80R (but not S134N) – in addition to the dimer interface mutant I149T, showed greater amplitudes of fluorescence change and shorter lag times than WT ( and ). In contrast, no enhancement was observed for the beta barrel mutants and the dimer interface mutant A4V at this guanidine concentration. In 2 M guanidine, on the other hand, all the mutants except S134N and D125H formed more amyloid than WT, with the greatest yield of amyloid observed for the “wild-type-like” beta barrel mutants E100G, G93A, and L38V ( and ). For the metal-binding-region mutants, the stimulation of amyloid formation at pH 5 is likely due to the fact that these mutations allow dissociation of metals from the enzyme at a higher pH than is observed for WT SOD1. For the wild-type-like mutations the stimulation of amyloid formation at pH 5 may be due to increased susceptibility of the mutants to acid-induced unfolding.
Amyloid formation from WT SOD1 and selected mutant SOD1 proteins at pH 5.
The enhancement of amyloid formation was manifested as a greater amplitude of ThT fluorescence and/or a reduced lag time. The amplitude of ThT fluorescence must be interpreted cautiously, and it is possible that some of the increase could be due to formation of an aggregate with a greater affinity for ThT, rather than a greater amount of aggregate. While mutants that showed both a greater fluorescence amplitude and a shorter lag time clearly enhance amyloid formation, those cases where the increased fluorescence was accompanied by a lag time equal to that of WT (e.g. L38V in 2 M urea) or even greater than that of WT (E100G in 2 M urea) are difficult to explain, and in these cases the evidence for an enhancing effect of the mutation on amyloid formation is not as strong.
Effect of Mutations on Fibrillation of Apo-SOD1 at pH 7
Apoproteins with a variety of ALS mutations (A4V, C146R, E100G, G85R, H46R, and I113T) formed amyloid at pH 7 in the presence of 1 M guanidine; no consistent effect of the mutations was observed, with some mutations forming more amyloid and others less amyloid than WT SOD1. In the absence of guanidine most of the mutations appeared to reduce the yield of amyloid, with the A4V mutation completely abolishing amyloid formation (Table S1
). However, electron microscopy of apo-A4V incubation mixtures revealed a variety of amorphous aggregates (data not shown), suggesting that A4V promoted the formation of amorphous aggregates instead of fibrillar aggregates. The possible existence of multiple aggregation pathways, some leading to amyloid and some to amorphous aggregates, both containing intermolecular disulfide bonds (), make the effects of mutations on apo-SOD1 amyloid formation difficult to interpret.
The situation was quite different for SOD1 mutants lacking free cysteine. No amyloid was observed for apo-AS (WT) SOD1 (), even after two weeks of incubation. On the other hand, apo-AS/A4V and AS/G93A formed amyloid readily, and a very small amount of amyloid was produced from apo-AS/G85R. These results indicate that when intermolecular disulfide formation is prevented by the absence of free cysteines, only metal-free SOD1 proteins bearing certain ALS mutations can form amyloid at neutral pH. Similar results were obtained by DiDonato et al. 
for the incubation of AS SOD1 and SOD1 proteins bearing mutations in an AS background at pH 3.5.
Aggregation assays with mutant SOD1 lacking free sulfhydryl groups.
Effect of Mutations on Fibrillation of Metalated SOD1 in 0.1 M TCEP at pH 7
In the presence of 0.1 M TCEP, nearly all FALS mutants examined showed enhanced amyloid formation compared to WT SOD1, and in a number of cases (C146R, H46R, and H80R) the lag time was substantially reduced ().
Kinetic parameters for amyloid formation from metalated SOD1 at pH 7 in the presence or absence of TCEP.
The ALS mutant C146R, which lacks the 57-146 intramolecular disulfide bond, produced a slightly lower yield of amyloid than WT SOD1, but the lag time was much shorter. The kinetics were similar whether or not TCEP was present, as would be expected, since TCEP should have no effect on a protein which lacks disulfide bonds. The result with C146R indicates that the absence of the intramolecular disulfide bond is sufficient to allow SOD1 to form amyloid. The only other SOD1 protein that formed amyloid at pH 7 even in the absence of either EDTA or TCEP was H80R, a mutant SOD1 which is nearly devoid of both copper and zinc () and therefore behaves like other apo-SOD1 proteins ().
In the presence of 0.1 M TCEP AS, AS/A4V, AS/G93A, and AS/G85R all showed a greater yield of amyloid formation relative to WT SOD1 ( and ). The lag time was much shorter than that of WT for AS/A4V, AS/G93A, and AS/G85R, but not for AS. The effect of removing cysteines 6 and 111 on the yield of amyloid, even in the absence of ALS mutations, is difficult to explain; the replacement of these two cysteines may either cause some destabilization of the tertiary structure of SOD1 
or promote the formation of amyloid instead of other types of aggregates, like amorphous aggregates.