Effect of Metal Loading and Subcellular pH on Net Charge of Superoxide Dismutase-1

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined—at any pH—for nearly all proteins. This study used lysine-acetyl “protein charge ladders” and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)—whose aggregation causes amyotrophic lateral sclerosis (ALS)—as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn2 + or Cu2 + ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~ 4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2–4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~ 3 units, instead of ~ 14) and did not undergo charge inversion at its isoelectric point (pI = 5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5–8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~ 45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization.