Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

作者：Shi Yunhua; Mowery Richard A; Ashley Jonathan; Hentz Michelle; Ramirez Alejandro J; Bilgicer Basar; Slunt Brown Hilda; Borchelt David R; Shaw Bryan F^*

来源：Protein Science, 2012, 21(8): 1197-1209.

DOI：10.1002/pro.2107

• lysine acetylation
• amyotrophic lateral sclerosis
• superoxide dismutase
• post-translational modification
• electrophoretic mobility
• surfactant
• lipid
• gel shifting
• phosphorylation

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).

• 出版日期2012-8