title: Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy.
abstract: Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.
authors.affiliation: Department of Chemistry and Biochemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
authors.forename: Pik K
authors.initials: PK
authors.lastname: Chan
authors.forename: Madhuri
authors.initials: M
authors.lastname: Chattopadhyay
authors.forename: Shivani
authors.initials: S
authors.lastname: Sharma
authors.forename: Puneet
authors.initials: P
authors.lastname: Souda
authors.forename: Edith Butler
authors.initials: EB
authors.lastname: Gralla
authors.forename: David R
authors.initials: DR
authors.lastname: Borchelt
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Joan Selverstone
authors.initials: JS
authors.lastname: Valentine
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D059748:Proteolysis; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D000072105:Superoxide Dismutase-1
chemical_list: D000682:Amyloid; D050505:Mutant Proteins; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
keywords: aggregation; mass spectrometry; neurodegeneration; protein misfolding