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

Val -3 Ala mutations within the effective transmembrane segment ofa model single-spanning membrane protein, the 50-residue major coat (gene VM) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilzation of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which induded those in which each of the four valine residues was replaced by an alanine residue. Sicant variations found among these Val -* Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their a-helix -* «-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (0) nonmutatable sites in the mutant protein library, (u) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iu) energyminimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential proteininteractive residues (V30, V31; G34, A35; G38, 139) and membrane-interactive residues (M28, V29; 132, V33; T36, 137). The overall results constitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.Although most transmembrane (TM) regions ofintegral membrane proteins are presumed to be a-helical in conformation, glycine and p,-branched residues (valine, isoleucine, threonine), known to disfavor a-helices in soluble proteins (1), often account for nearly 50%6 ofTM amino acids (2). As many membrane proteins require function-related conformational change(s) [e.g., during membrane insertion (3) or signaling (4)], the TM sequence-particularly through its glycine and (3-branched content-may also contribute to function (e.g., conformational flexibility and/or helix destabilization) at various stages of the protein life cycle (5) rather than serve exclusively to anchor the protein to the membrane. DNA (7, 8). In contrast, the transient existence of coat protein in the E. coli cytosol must favor a watersoluble conformation, in which nascent membrane proteins are likely to have a (3-sheet ("extended") conformation (9).The sequences ofa variety offilamentous phage coat proteins tend to be highly conserved, perhaps reflecting an inherent requirement for multiple conformations dictated by primary sequence during the phage life cycle (10).DNA-bound M13 coat proteins are essentially 100%o a-helical, while helical aggregates are ...

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