| Approximately 100 mutations to the antioxidant enzyme copper-zinc superoxide dismutase (SOD1) cause a debilitating, fatal neurodegenerative disease known as familial amyotrophic lateral sclerosis. The mutations are very diverse both in their location on the protein backbone and in their physical effects on the SOD 1 mutant protein. To study how these diverse mutations cause the same disease we generated a large subset of mutant SOD1 proteins by expressing and purifying them from yeast. The mutant proteins were subjected metal analysis, activity measurements and visible spectroscopy and it is clear from these data that there are at least two distinct groups of FALS mutants. One group is nearly indistinguishable from the wild type while the other group is consistently isolated with reduced levels of metals, exhibits different activities, and have altered visible spectra when compared to the wild type enzyme. We determined the three-dimensional structures of biologically metal-loaded wild type in 2 space groups, apo-wild type, A4V, L38V, H43R, H46R, G85R, G93A, E100G, I113T, D125H and S134N. FALS mutant SOD1 proteins L38V (2nd crystal form), H48Q, H80R (two crystal forms), G93A (2nd crystal form), G93V, D101N, L144F, L144S, A145T and I149T are near completion or have been completed recently. We are also interested in what factors influence the inherent stability of the dimer interaction of SOD L To investigate these factors, we employed the method of analytical ultracentrifugation to study the monomer-dimer interactions in solution. Sedimentation equilibrium and velocity experiments showed both metal-containing and apo-SOD1 was present in solution as a single dimeric species. Induced monomerization in the presence of increasing amounts of guanidine, and upon reduction of the intrasubunit disulfide bond enabled us to characterize the dimer interface in metal-containing and apo-wild type SOD1 proteins. This work sets the stage for similar work to be done using FALS mutants, a project currently underway in the Hart laboratory. Thioesterase activity of SOD1 was first discovered in our laboratory, but the determination of the active form of the protein and the nature of the active site has been elusive because the activity is only observed under specific conditions. Evidence suggested that activity was related to a specific metal species, so we used differential scanning calorimetry, and chemical modification of specific residues to show which metal species may be involved in the thioesterase activity and which residues might be involved. Based on the structures of well characterized thioesterases, we propose a possible site on SOD1 that may be involved in the thioesterase activity. |
