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  • Title: Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1.
    Author: Shi Y, Mowery RA, Shaw BF.
    Journal: J Mol Biol; 2013 Nov 15; 425(22):4388-404. PubMed ID: 23871896.
    Abstract:
    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 Zn(2+) or Cu(2+) 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.
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