2008 Eighth Annual Beckman Scholars Symposium
Friday Poster Session - July 28, 2006

Twenty percent of familial amyotrophic lateral sclerosis (fALS) cases are linked to mutations in Cu,Zn superoxide dismutase (SOD1). These mutations appear to increase the enzyme’s tendency to form insoluble aggregates in the motor neurons of ALS patients. SOD1 is a dimer of 16-kD chains stabilized by abundant hydrophobic interactions at the dimer interface. The initial steps in the aggregation pathway appear to be the loss of the structure-stabilizing zinc ion and the dissociation of the dimer into monomers. We have investigated the effects of ALS-related mutations on the dissociation of SOD1 dimers to monomers by measuring the fluorescence polarization due to each monomer’s single tryptophan residue. Guanidine titrations of metallated SOD1 variants at pH 7 and room temperature show that the mutations increase the sensitivity of the dimer to guanidine-induced dissociation. The guanidine concentration required for half-dissociation was 5.5 M for WT SOD1 but was reduced to about 2 M for the “WT-like” mutants G93A and A4V and to about 1 M for the metal-binding region (MBR) mutants G85R and H46R. WT apo-SOD1 was half-dissociated at 1 M guanidine, and mutant apo-SOD1’s at concentrations of 0.5 M. Dimer-monomer dissociation constants of apo-SOD1 variants were determined by measuring the dependence of fluorescence polarization on protein concentration at pH 7 and 37°C. Apo-WT has a dissociation constant of 5 x 10-7 M, whereas WT-like mutants have dissociation constants an order of magnitude higher (4 x 10-6 M for apo-G93A and 5 x 10-6 M for apo-A4V). Thus, SOD1 mutations increase the enzyme’s tendency toward dissociation by weakening hydrophobic interactions at the dimer interface. This effect would be expected to make a substantial contribution to the increased tendency of ALS-related mutant SOD1 variants to aggregate.