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

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76706, USA.
Protein Science (Impact Factor: 2.85). 08/2012; 21(8):1197-209. DOI: 10.1002/pro.2107
Source: PubMed


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 ~80-101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80-101 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 80-101 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).

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    • "The G85R variant, which possess the same type of charge shifting mutation, does not produce the same effect (Fig. 2, lane 5). However, it is well established that the G85R variant exhibits aberrant electrophoretic mobility in SDS-PAGE [36] and we noted that the G85R variant produced a faster migrating entity on the BN-gels (Fig. 2, lane 5, asterisk). In cells expressing the G85R variant we observed a strongly reactive entity that migrated slower than WT-hSOD1 (2 asterisks) and could be an assembly of SOD1-G85R that is larger than a homodimer. "
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