Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

TLDR

Gel shifting, the anomalous migration of membrane proteins on SDS‑PAGE, is widespread and may arise from altered detergent solvation that replaces protein‑detergent contacts with protein‑protein interactions, linking detergent binding to folding. The study investigates how helical membrane proteins, specifically hairpin sequences from CFTR transmembrane segments 3 and 4, exhibit anomalous gel mobility. The authors use a library of wild‑type and mutant CFTR hairpin sequences, including disease‑phenotypic substitutions, to probe this behavior. Hairpins migrated 10–30 % faster or slower than predicted, loaded 3.4–10 g SDS per g protein, and mutant gel shifts correlated strongly with SDS loading capacity (R² = 0.8) and helicity (R² = 0.9), indicating that altered detergent binding drives gel shift, that the V232D mutant may impair CFTR function via disrupted protein‑lipid interactions, and that detergent binding could serve as a rapid screen for membrane proteins with stable tertiary or quaternary structures.

Abstract

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.