Abstract 1351 The N53D Deamidation Mimic Causes Misfolding and Aggregation of Superoxide Dismutase 1

The toxic effects of Superoxide Dismutase 1 (SOD1) aggregates on motor neurons and their contribution to the phenotype of amyotrophic lateral sclerosis (ALS) have been well documented. Multiple missense mutations resulting in the conversion of an asparagine residue into an aspartic acid residue have been discovered to cause familial ALS, raising the question of a potential, chemically equivalent, post-translational deamidation event in cases of sporadic ALS. Asparagine residue 53 in the SOD1 sequence has been computationally predicted to not undergo spontaneous deamidation; however our lab measured this deamidation in SOD1 extracted from human stem cell derived motor neurons. N53 is of particular interest due to its location in the zinc binding loop of SOD1's tertiary structure and proximity to a structure stabilizing disulfide bridge. This study aims to characterize the structural and behavioral properties of N53 deamidation mimic, N53D, to understand its implications towards SOD1 aggregation and, on a larger scale, the propagation of the sporadic ALS phenotype. Site directed mutagenesis was utilized to incorporate a single base pair change in the SOD1 sequence which was overexpressed in E. Coli. Size exclusion chromatography was used both as the final step of purification, isolating SOD1 homodimer, and as a method capable of calculating the relative occurrence of monomeric and dimeric SOD1. A larger occurrence of monomeric SOD1 was present in N53D samples, indicating the mutant is more prone to failures in dimerization and exists more frequently in an monomeric state of reduced stability that is highly susceptible to misfolding and aggregation. Thermal melts via circular dichroism conducted on purified dimer pools revealed N53D SOD1 begins to unfold more severely and at earlier temperatures than non-mutated SOD1. Thioflavin T assays quantified the amount of amyloid-like oligomers present in dimer populations upon biological temperature incubation and indicated N53D SOD1, incubated with its copper cofactor first, to have roughly twice the amyloid content of its WT counterpart. Spontaneous deamidation at site 53 in the SOD1 sequence is likely to result in misfolded and structurally unstable protein species that have an increased propensity for toxic aggregation. Through this mechanism, the N53D mutation could be one factor responsible for the propagation of degenerative symptoms in cases of sporadic ALS. This research has been funded by the ASBMB foundation through the Undergraduate Research Award and by Wesleyan University through the Summer Research In the Sciences Program.