Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.
Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.
Rath
Arianna
A
Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Glibowicka
Mira
M
Nadeau
Vincent G
VG
Chen
Gong
G
Deber
Charles M
CM
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D050505:Mutant Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D015203:Reproducibility of Results
C505032:CFTR protein, human; D003902:Detergents; D008565:Membrane Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D019005:Cystic Fibrosis Transmembrane Conductance Regulator