Direct biophysical comparisons of yeast and human copper-zinc superoxide dismutases: Dissecting the roles of metal binding in the protein maturation pathway

Copper-zinc superoxide dismutase (SOD1) is one of the most scrutinized metalloproteins; yet much remains unknown about this ubiquitous protein. For example, the steps on the pathway from the freshly transcribed polypeptide to the fully folded and active enzyme are not delineated. In order to be fully active, SOD1 must fold, obtain metals, oxidize the intramolecular disulfide, and dimerize, in an order and by mechanisms that remain mostly elusive. It is critical to understand these processes because mutations in the protein cause amyotrophic lateral sclerosis. This disease, now commonly thought to be a protein misfolding disease, may involve immature forms of the protein. In this dissertation, I examine post translational protein folding events and modifications and how they relate to overall stability of the SOD1 protein.; Human and yeast SOD1 proteins, the proteins of focus in this study, were isolated and characterized biophysically. Significant differences were observed in the overall stabilities of the apo proteins, where the hSOD1 protein was much more stable than ySOD1. We propose that these differences may be responsible for differences in the dependence on the copper chaperone for SOD1 (CCS) for activation in vivo. ITC was used to determine the thermodynamics of zinc binding to the apo proteins and again, large differences were observed in the metal affinities between the yeast and human SOD1 proteins. The ITC experiments on ySOD1 revealed that the dimer was destabilized compared to that of hSOD1.; A mutant of hSOD1, S,L 142, 144, P,P hSOD1, which was shown to mimic ySOD1 in vivo in terms of its requirement for CCS in vivo, was also isolated and characterized. In vitro metal titrations monitored spectroscopically revealed metal binding sites that more closely resembled yeast SOD1 protein. This protein was also determined to be overall less stable than hSOD1. The zinc binding site of the hSOD1 protein was also probed by engineering the site into a Type 1 blue copper site. A non-native cysteine residue incorporated into the zinc binding site produced an intense LMCT upon copper binding that is shown to be a very sensitive probe to the zinc site geometry.