Abstract
The protein superoxide dismutase 1 (SOD1) is a homodimeric cytosolic antioxidant protecting the cell against damage from superoxide. Prion-like misfolding of SOD1 is associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), leading to interest in the folding dynamics of SOD1. Previous work suggested that SOD1 folding/unfolding occurs in a three-state process: first the dimer monomerizes, then each monomer subunit folds/unfolds in a two-state process. Here we re-examine SOD1 folding at the single-molecule level, using force spectroscopy with optical tweezers. We focus on SOD1 in the reduced, unmetallated (apo) form, which is the least stable form and thus thought to be most relevant for misfolding. Force-extension curves (FECs) of the unfolding and refolding of isolated monomers reveal multiple intermediate states, showing that the folding is much more complex than expected: a variable number of intermediates is seen in different FECs, from 1 to 5. Examining dimeric SOD1, similar complexity is again observed, with 2 to 5 intermediates. Comparing the dimer to the monomer suggests that the dimerization enhances the cooperativity somewhat. Notably, occasionally the FECs reveal length changes that do not match the length expected for native folding, suggesting that they represent misfolding events.
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