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首页> 外文期刊>Journal of Molecular Biology >Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1
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Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1

机译：金属负载和亚细胞pH对超氧化物歧化酶-1净电荷的影响

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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.
机译：假定折叠蛋白质的净电荷会影响无数生化过程（例如蛋白质错误折叠，电子转移，分子识别）;但是，几乎没有测量净电荷的工具，而且对于几乎所有蛋白质，在任何pH值下，这种难以捉摸的性质都无法确定。这项研究使用赖氨酸-乙酰基的“蛋白质电荷阶梯”和毛细管电泳来测量超氧化物歧化酶-1（SOD1）的净电荷（其聚集导致肌萎缩性侧索硬化症（ALS））与金属离子和pH的函数关系。 apo-SOD1的净负电荷与预测值相似;然而，单个Zn2 +或Cu2 +离子的结合使净负电荷减少的幅度比预期的大（即〜4个单位，而不是2个单位），而SOD1蛋白在结合2-4个金属离子时经历电荷调节。从pH5到pH8（即与SOD1的多个亚细胞基因座一致的范围），holo-SOD1蛋白的净负电荷波动小于预期（即〜3个单位，而不是〜14个单位），并且未进行充电在其等电点（pI = 5.3）反转，但仍为阴离子。 SOD1净电荷沿着其金属键合途径以及整个溶剂pH值的调节，可洞察其金属诱导的成熟和酶活性（在pH5-8范围内仍受扩散限制）。整个亚细胞pH值中holo-SOD1的阴离子性质表明，与亚细胞定位相关的〜45种不同的ALS连锁突变会降低SOD1的净负电荷。

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