Abstract
Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Here we study the most misfolding-prone form of SOD1, reduced un-metallated monomers, using optical tweezers to measure unfolding and refolding of single molecules. We find that the folding is more complex than suspected, resolving numerous previously undetected intermediate states consistent with the formation of individual β-strands in the native structure. We identify a stable core of the protein that unfolds last and refolds first, and directly observe several distinct misfolded states that branch off from the native folding pathways at specific points after the formation of the stable core. Partially folded intermediates thus play a crucial role mediating between native and non-native folding. These results suggest an explanation for SOD1’s propensity for prion-like misfolding and point to possible targets for therapeutic intervention.
Highlights
Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease amyotrophic lateral sclerosis (ALS), but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly
Monomeric human SOD1, mutated to remove solvent-exposed cysteines[20, 21] and disrupt the dimer interface[20, 21, 56], was attached to DNA handles bound to polystyrene beads held in dual-trap tweezers (Fig. 1b), and the extension of the protein–DNA construct was measured as the traps were moved apart and back together at constant speed to ramp the force up and down
Unfolding force–extension curves (FECs) showed a non-linear rise of force with extension—as is typical for polymers stretched under tension—interrupted by one or more “rips” characteristic of unfolding transitions, resulting in sawtooth patterns in the FECs (Fig. 1c, black)
Summary
Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Folded intermediates play a crucial role mediating between native and non-native folding These results suggest an explanation for SOD1’s propensity for prion-like misfolding and point to possible targets for therapeutic intervention. Native structure is generally believed to form via a three-state process, with each monomer subunit folding in a two-state process before dimerization[20,21,22,23,24], some studies have suggested a more complex process involving intermediates[25,26,27]. The structural properties of misfolded forms of SOD1 have begun to be studied[34, 35], but the nature of the misfolding mechanism, the role of intermediate states (if any), and the identity of the toxic species all remain controversial[24, 27, 30].
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