Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding
Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding
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DOI：
10.1002/pro.2107
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发表时间：
2012-08-01
期刊：
影响因子：
8
通讯作者：
Shaw, Bryan F.
中科院分区：
文献类型：
--
作者：
Shi, Yunhua;Mowery, Richard A.;Shaw, Bryan F.
胞浆蛋白的氨基酸取代或翻译后修饰可能会导致 SDS-PAGE 期间其电泳迁移率发生不可预测的变化。几十年来，这种类型的凝胶转移一直困扰着生物化学家和生物学家。我们确定了一种凝胶转移机制，该机制在一组 ALS（肌萎缩侧索硬化症）突变 hSOD1（超氧化物歧化酶）蛋白、翻译后修饰的 hSOD1 蛋白和来自不同生物体的同源 SOD1 蛋白中占主导地位。通过首先比较整个 hSOD1 中的 39 个氨基酸取代如何影响 SDS-PAGE 迁移，我们发现导致凝胶迁移的取代发生在单个多酸结构域（残基 80101）内，并且是非等电的。降低结构域 80101 净负电荷的取代增加了迁移；只有一种替代增加了净负电荷并减缓了迁移。毛细管电泳、圆二色性和尺寸排阻色谱法证明，氨基酸取代通过促进三到四个额外 SDS 分子的结合来增加 SDS-PAGE 期间的迁移，而不会显着改变 hSOD1-SDS 复合物的二级结构或斯托克斯半径。 SOD1 凝胶迁移需要结构域 80101 的高负电荷：中和多酸结构域（通过嵌合的小鼠-人 SOD1 融合蛋白）可抑制氨基酸取代引起的凝胶迁移。这些结果表明，突变胞质蛋白的凝胶移动模式可用于：（i）识别一级结构中控制变性胞质蛋白和 SDS 之间相互作用的结构域，以及（ii）识别相互作用的主要化学机制（例如，疏水性与静电）。
The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of gel shifting has perplexed biochemists and biologists for decades. We identify a mechanism for gel shifting that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues 80101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).