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.detergent binding ͉ gel shifting ͉ membrane proteins ͉ SDS-PAGE ͉ sodium dodecyl sulfate

Membrane proteins adopt two fundamental types of folds in nature: membranes in all organisms harbor α-helical bundles linked by extramembranous loops of varying length, while β-barrel structures are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Here we report that turn-inducing loop mutations in a transmembrane hairpin induce the conversion of an α-helical hairpin to β-sheet oligomers in membrane environments. On the basis of an observation of a sequence bias toward Pro and Gly in the turns of native β-barrel membrane proteins, we characterized in sodium dodecyl sulfate (SDS) micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers several "hairpin" constructs of cystic fibrosis transmembrane conductance regulator transmembrane segments 3 and 4 (TM3-loop-TM4; loop region being (215)IWELLQASA(223)) in which Pro-Gly residues were either inserted or substituted at several positions. Remarkably, suitable positioning of the Pro-Gly doublet caused the adoption of stable β-sheet structures by several mutants in SDS micelles, as shown by circular dichroism spectroscopy, concurrent with a ladder of discrete oligomers observed via SDS-polyacrylamide gel electrophoresis. Reconstitution of wild-type (WT) TM3/4 into POPC vesicles studied by Trp fluorescence, in conjunction with positional quenchers in brominated phospholipids, indicated a transbilayer position for helical WT TM3/4, but likely a largely surface-embedded conformation for the β-sheet mutant with loop region IWPGELLQASA. To the best of our knowledge, such a complete change in the fold with a minimal number of mutations has not been previously observed for a membrane protein. These facile α-helix to β-sheet conversions highlight the contribution of loops to membrane protein structure.