title: Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798-7348, USA.
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Shi
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mowery
authors.forename: Bryan F
authors.initials: BF
authors.lastname: Shaw
mesh_terms: D000107:Acetylation; D002021:Buffers; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry*; D017510:Protein Folding; D055672:Static Electricity; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D002021:Buffers; D008670:Metals; C000606290:SOD1 protein, human; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc
keywords: ALS; CE; ICP-MS; SOD1; TCEP; WT; amyloid; amyotrophic lateral sclerosis; capillary electrophoresis; copper; inductively coupled plasma-mass spectrometry; metal ions; protein misfolding; superoxide dismutase-1; tris(2-carboxyethyl)phosphine; wild-type; zinc