Equilibrium Thermodynamic Analysis of Amyotrophic Lateral Sclerosis-Associated Mutant Apo Cu,Zn Superoxide Dismutases†,‡

The folding and thermodynamic properties of metal free (apo) superoxide dismutases (SODs) are systematically analyzed using equilibrium guanidinium chloride (GdmCl) curves and differential scanning calorimetry (DSC). Chemically and structurally diverse amyotrophic lateral sclerosis (ALS)-associated mutations (G85R, G93R, E100G, I113T) are introduced into a pseudo-wild-type background that has no free cysteines, resulting in highly reversible unfolding. Analysis of the protein concentration dependence of GdmCl curves reveals formation of a monomer intermediate in equilibrium with native dimer and unfolded monomer. Global fitting of the data enables quantitative measurement of free energy changes for both dimer dissociation and monomer intermediate stability. All the mutations decrease protein stability, mainly by destabilizing the monomer intermediate, but also by tending to weaken dimerization, even for mutations far from the dimer interface. Thus, the effects of mutations seem to propagate through the ap...