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The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H͞D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6°C to 56.2°C, i.e., similar to or higher than that of the WT apoprotein (Tm ‫؍‬ 52.5°C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0°C to 52.4°C. The H͞D exchange properties of the mutants were also wideranging: the MBR mutant apoproteins exhibited H͞D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H͞D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H͞D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.differential scanning calorimetry ͉ hydrogen-deuterium exchange ͉ protein stability ͉ protein aggregation ͉ neurodegenerative disease P rotein misfolding and aggregation have been linked to many diseases, including Alzheimer's disease, cystic fibrosis, transmissible spongiform encephalopathies, and ALS, but the pathways followed by pathogenic proteins from translation to disease-causing states are not completely understood (1-3). In some cases, partial or complete unfolding from the native state precedes protein aggregation, and thus the stability of a protein's native state may provide one measure of its propensity to aggregate. However, many familial protein misfolding diseases are caused by proteins that are not destabilized relative to their WT counterparts (4-6), implying that additional intrinsic or extrinsic factors may be required for protein aggregation.Our recent studies of a large number of ALS-mutant Cu,Znsuperoxide dismutase (SOD1) proteins have revealed that there is great diversity in the biophysical properties of these proteins (7-12). In contrast, Lindberg et al. (13) reported in 2002 that instability of the apoproteins of ALS-mutant SOD1 proteins is a ''common denominator'' among the nearly 100 known ALSlinked SOD1 mutations. More recently, Furukawa and O'Halloran (14) have reported that some of the destabilized mutant apoproteins studied by Lindberg et al. are further destabilized when the intrasubunit disulfide bond is reduced, again suggesting that protein destabilization is ...

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Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.