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

      Protein Science : A Publication of the Protein Society

      metabolism, Surface-Active Agents, genetics, analysis, Superoxide Dismutase, Protein Structure, Tertiary, Protein Processing, Post-Translational, Protein Binding, Mutant Proteins, Motion, Mice, chemistry, Lysine, Humans, methods, Electrophoresis, Polyacrylamide Gel, Animals, Amyotrophic Lateral Sclerosis, Amino Acid Substitution, Acetylation

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          Abstract

          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). Copyright © 2012 The Protein Society.

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          Author and article information

          Journal
          10.1002/pro.2107
          3537240
          22692797

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