Mutations in copper-zinc superoxide dismutase (SOD1) are known to cause amyotrophic lateral sclerosis by an unknown molecular mechanism. At this time, there are over 110 mutations in the SOD1 primary sequence that give rise to the same clinical pathology. These mutations are primarily single amino acid substitutions, however, some are insertions, deletions, or C-terminal truncations. Despite the fact that the set of ALS-causing SOD1 mutant proteins is biophysically diverse, there is a common cytotoxic property shared by this class of proteins. This toxic property is likely to be related to an increased propensity for these proteins to form soluble and/or insoluble aggregates which prove to be toxic to motor neurons. In this dissertation I examine the solution dynamics of these SOD1 mutant proteins using hydrogen/deuterium exchange (HDX).

A series of ALS-causing SOD1 mutant proteins were isolated from a yeast expression system and characterized using both global and site-specific HDX. Our HDX studies primarily focused on the apo forms of these proteins, since this form is likely to represent the most aggregation prone state. In the case of apo A4V SOD1, we discovered that this protein features more solvent-accessible domains than human wild-type (hWT) SOD1 does. In addition, apo A4V undergoes a slow local unfolding process that does not occur in hWT. Also, our studies revealed that in its disulfide-reduced form, apo A4V exchanges as if it were a random-coil polypeptide–in contrast, the corresponding form of hWT is a structured protein.

In order to understand better the maturation process that occurs with hWT SOD1, we used HDX to examine the dynamics of hWT at different stages of maturity (apo and disulfide-reduced, metallated and disulfide-reduced, and metallated and disulfide-intact). Our studies revealed that the coordination of metals to the disulfide-reduced apoprotein produces profound changes in the dynamical signatures of the protein in all regions for which sequence coverage is afforded. To our surprise, we learned that the subsequent formation of the disulfide bond (with metals present) causes only very subtle changes in the HDX behavior of all the covered regions of the protein. Most notably, we observed that the dynamics of the metallated protein changes only very slightly even in regions near the disulfide bonded cysteine residues.