Pepsin digest coverage map of SOD1. Top panel, bold lines indicate the peptide fragment ions identified. Note the redundant coverage (two or more peptide ions/amino acid) of the entire SOD1 primary sequence. Bottom panel, H/D exchange of WT copper/zinc SOD1 at 4 °C (in triplicate). From top, the results shown were measured in March, April, and January. Each block contains four time points 15, 50, 150, and 500 s. The deuteration level at each time point of each peptide is color-coded as a heat map (shown to the right).

WT (left panel) versus fALS mutant G85R (right panel) superoxide dismutase showing percentage deuteration levels. Percentage deuteration levels following 15 s of exchange were threaded onto a human copper/zinc-SOD crystal structure, Protein Data Bank code 1SPD (1). The region comprising the dimeric interfaces is the vertical blue area in the center of the structure. Red indicates the regions which exchange fast (therefore dynamic), and blue indicates the regions which exchange slowly (therefore rigid). The data demonstrate that the region corresponding to the dimer interface (labeled in structure) affords the greatest protection from amide H/D exchange, whereas the electrostatic loop (labeled in structure) affords the least protection.

Changes to the structure/dynamics of the electrostatic loop are a shared property of fALS SOD1 mutants. Deuterium incorporations relative to copper/zinc-WT SOD1 (Fig. 2 illustrates absolute rates) at times (from top to bottom in the matrix) 15, 50, 150, and 500 s were compared for 13 fALS SOD1 variants, copper-deficient SOD1, and a duplicate copper/zinc SOD1 control. WT-like variants (upper eight rows) and metal-binding variants (lower five rows) are separated by a larger space. Note that each mutant was found to increase the dynamics of the electrostatic loop. This region of shared perturbation was colored orange and threaded onto the secondary structure (middle panel), where the thickness of orange is proportional to the number of mutants affected in a particular region, and onto human copper/zinc-SOD crystal structure (bottom panel; Protein Data Bank code 1SPD) (1) with mutated residues labeled. Note that the dimer interface is predominantly the N terminus, residues 49–54, 113–115, and 148–153 and is shown as black bars. In triplicate experiments, no difference greater than 5% was observed between WT SOD1 controls.

Effect of metallation on structure/dynamics of SOD1. Shown is the correlation between metal content and fALS SOD1 variant H/D exchange perturbations with respect to wild type SOD1. a, copper content and H/D exchange perturbations at electrostatic loop residues 119–132; b, correlation between zinc content and H/D exchange perturbations at zinc loop residues 56–96; c, correlation between zinc content and H/D exchange perturbations at electrostatic loop residues 119–132. R² value of linear fits are 0.25 (a), 0.75 (b), and 0.43 (c).

Correlation between loop structure/dynamics and overall SOD1 structure/dynamics. a, correlation between fALS SOD1 variant H/D exchange perturbations for zinc loop (average of % deuteration increase compared with WT for the peptide covering residues 56–96) versus sum of total exchange; b, correlation between fALS SOD1 variant H/D exchange perturbations for electrostatic loop (sum of average of % deuteration increase compared with WT for peptides covering residues 119–132, 119–144, and 133–144) versus total exchange. For S134N, peptide 119–132 was not observed, and so deuteration of 119–144 was taken as the value for residues 133–144 and 119–132. R² and p values of linear fits are 0.80 and p < 0.0001 (a) and 0.84 and p < 0.0001 (b).