Amyotrophic lateral sclerosis (ALS) is a fatal, autosomal dominant, progressive degenerative disease of motor neurons [4]. The inherited form, familial ALS (FALS), represents approximately 5-10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper-zinc superoxide dismutase (SOD1) [5, 6]. To date, approximately 100 distinct mutations, most of which result in single amino acid substitutions, have been identified. Although the molecular basis for SOD1-mediated FALS has remained obscure, aggregates containing pathogenic SOD1 proteins are observed in spinal cord neurons of FALS patients and in transgenic mouse models of the disease [7-9]. Formation and/or accumulation of these SOD1 - containing aggregates is now widely believed to reflect the "toxic gain-of-function" ascribed to pathogenic SOD1, although the exact mechanism through which they exert their toxic effects remains unclear. The spatial distribution of FALS SOD1 mutations on the 3-D scaffold of the protein is broad, falling into two categories we term "metal-binding region" (MBR) and "wild type-like" (WTL) mutants [10]. Our previous crystallographic studies on five members of the MBR mutant class of SOD1 reveal that they are metal deficient. The absence of metal ions leads to conformational changes in loop elements that deprotect the edge strands of beta-sheets in the protein. This loss of protection in turn gives rise to a "gain-of-interaction" (GOI) between mutant SOD1 dimers that promotes the formation of linear and helical filamentous arrays [1]. Thus, conformational rearrangement in the metal-deficient enzyme leading to higher order oligomeric assemblies could represent the toxic property common to mutants of SOD1 linked to FALS. Some members of the WTL mutant class of SOD1 are so destabilized in their apoprotein forms that they may aggregate before they ever have a chance to be metaHated or dimerize properly [11]. However, there are other WTL mutants that are just as stable as WT SOD1 in both their metallated and apo- forms (see Preliminary Results, Project 1). One possible explanation for this apparent contradiction is that the latter WTL mutant SOD1 proteins are not properly metallated in vivo due to abnormal interactions with metallochaperones. Another possibility is that these mutants are more easily oxidatively damaged in vivo and that the oxidized pathogenic protein suffers an impairment in its metal binding ability and thus is more susceptible to misfolding and/or aggregation. The goals of this research project (Project 2) are to derive a better understanding of the mechanisms of aggregation of pathogenic SOD1 through the use of a wide range of complementary biophysical methods and to couple this information with that gained from our collaborative efforts to probe the determinants of aggregation of these proteins in vivo. This fundamental understanding of pathogenic SOD1 aggregation is a prerequisite for future efforts aimed at obtaining therapeutic agents for the disease that target protein misfolding and/or protein degradation pathways.