Immature ALS-associated mutant superoxide dismutases form variable aggregate structures through distinct oligomerization processes.

Protein misfolding and aggregation are hallmarks of many diseases, including amyotrophic lateral sclerosis (ALS). In familial ALS, aberrant self-association of mutant Cu,Zn-superoxide dismutase (SOD1) is implicated as a key contributor to disease. Mutations have the largest impacts on the stability of the most immature form of SOD1, the unmetallated, disulfide-reduced monomer (apoSH SOD1). Here we demonstrate that, despite the marginal stability of apoSH SOD1, aggregation is little correlated with the degree of protein unfolding, and multiple modes of aggregation occur, depending on the mutation and solution conditions. Light scattering and atomic force microscopy reveal two distinct mutant SOD1 behaviours: high aggregator mutants form abundant small assemblies, while low aggregator mutants form fewer, more fibre-like aggregates. Attenuated total reflectance-Fourier transform infrared spectroscopy and Thioflavin T binding show the aggregates maintain native-like anti-parallel beta structure. These results provide new evidence that ALS-associated mutations promote the aggregation of apoSH SOD1 through multiple pathways, with broad implications for understanding mechanisms of protein self-association in disease and biotechnology.