Thermal stability, catalytic activity and spectroscopic properties of amyotrophic lateral sclerosis-associated copper-zinc superoxide dismutases

Copper-zinc superoxide dismutase (SOD1) catalyzes the disproportionation of superoxide. Over 100 distinct mutant forms of SOD1 cause the fatal neurodegenarative disease Amyotrophic Lateral Sclerosis (ALS). One objective of the investigations in this dissertation was to determine the features of mutant proteins that may be responsible for causing ALS. To achieve this objective the biophysical and biochemical properties of a large number of mutant proteins were determined and compared to those of the WT.; Chapter 2 and 3 detail a comparison of the metal ion binding properties, SOD activity, and electronic spectroscopy of WT and 60 mutants. These studies led to the identification of two classes of mutants we termed "WT-like" and "metal-region-binding". The WT enzyme and WT-like mutants exhibited virtually identical properties while the properties of metal-binding-region mutants were notably different.; It has been hypothesized that a decrease in structural stability is relevant to the toxic properties of the mutant SOD Is. This hypothesis was tested by investigating the thermal stability of different metallated forms of the WT enzyme (Chapter 5) and of SOD I mutants from both groups in their as-isolated form (Chapter 4), and in their metal-free (apo) form (Chapter 6). These studies led to the elucidation of two distinct heat induced protein unfolding pathways and to the assignment of endotherms arising from different metallated forms of the WT enzyme. We also found that the as-isolated WT-like mutants were all destabilized compared to the corresponding forms of the WT SOD1. However, the degree of destabilization was more pronounced in their apo-form. The stability of the as-isolated metal-binding-region mutants could not be ascertained due to their low metal content. Their apo forms, however, exhibited an equal or greater stability compared to the apo WT SOD 1. Combined, the data in chapters 2--6 indicate that the decreased stability of the apo proteins, relative to their metallated forms, may play a role in causing ALS.