Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding.
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).
Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76706, USA.
Yunhua
Y
Shi
Richard A
RA
Mowery
Jonathan
J
Ashley
Michelle
M
Hentz
Alejandro J
AJ
Ramirez
Basar
B
Bilgicer
Hilda
H
Slunt-Brown
David R
DR
Borchelt
Bryan F
BF
Shaw
D000107:Acetylation; D019943:Amino Acid Substitution; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009038:Motion; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013482:Superoxide Dismutase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D013501:Surface-Active Agents / Q000378:metabolism*
D050505:Mutant Proteins; C000606290:SOD1 protein, human; D013501:Surface-Active Agents; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D008239:Lysine