Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding
| Autor: | Bryan F. Shaw, Alejandro J. Ramirez, Michelle Hentz, Richard A. Mowery, Jonathan D. Ashley, Hilda Slunt-Brown, Yunhua Shi, Basar Bilgicer, David R. Borchelt |
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| Rok vydání: | 2012 |
| Předmět: |
Circular dichroism
Plasma protein binding Biochemistry Mice Motion Surface-Active Agents Superoxide Dismutase-1 Protein structure Animals Humans Molecular Biology Protein secondary structure Polyacrylamide gel electrophoresis chemistry.chemical_classification Superoxide Dismutase Lysine Amyotrophic Lateral Sclerosis Protein primary structure Acetylation Articles Fusion protein Protein Structure Tertiary Amino acid Amino Acid Substitution chemistry Electrophoresis Polyacrylamide Gel Mutant Proteins Protein Processing Post-Translational Protein Binding |
| Zdroj: | Protein Science. 21:1197-1209 |
| ISSN: | 0961-8368 |
| DOI: | 10.1002/pro.2107 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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