Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

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.

Fig. 1.

Hairpin sequences and SDS-PAGE analysis. (A) Amino acid sequence of the WT TM3/4 hairpin. Residues predicted to be in helical (green text) or loop (black text) regions of CFTR are shown (34). Residues substituted in this work are underlined. (B) Representative SDS-PAGE of helical hairpin mutants. Positions of MW standards (in kDa) are indicated. This panel is a composite of two gels, as indicated by the solid line between lanes. PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively.

Fig. 2.

CD profiles for all hairpins in 0.3% SDS and 50 mM sodium phosphate, pH 7. Mutants are arranged (right of panel) in order of increasing helicity at 222 nm. PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Spectra shown are the average of 3–6 independent experiments. See Table 2 for a list of helicity values.

Fig. 3.

Correlation of SDS binding and PAGE mobility. The relationship between bound SDS and gel shift is shown with the correlation coefficient (R²) of the best fit line. Trendline P value is 0.001. The variations in mutant-WT values were propagated from the standard deviations of each mean using standard formulae.

Fig. 4.

Correlation of SDS binding and hydropathy. Mutation-dependent changes in bound SDS are plotted as a function of hydropathy changes. Mutant-WT hydropathy values were calculated individually with 6 common scales (–40) and the results averaged, such that positive hydropathy indicates increased apolarity and negative hydropathy increased hydrophilicity. The E217S/S222E mutant is marked with an asterisk. Correlation coefficients (R²) of the best fit line in the absence of E217S/S222E (red) or with all mutants (black) are shown. Trendline P value in the absence of E217S/S222E is 0.002.

Fig. 5.

Interrelationship between hairpin conformation and detergent binding. Hairpins (yellow) loaded with SDS molecules (blue) are represented. The number of SDS molecules on each hairpin, and the number of turns of helical structure, are intended to illustrate relationships between relative levels of detergent binding and/or helicity in a quantitative manner. The necklace and bead structure typical of an unfolded membrane protein with its TM segments fully coated with detergent acyl chains (A) is shown at the center. Potential alterations in SDS loading accompanying hydropathy reductions (B–D) are shown at the top; in some instances, regions with reduced hydropathy may no longer intercalate with lipid acyl chains but instead may partition closer to the micelle surface (B) or remain uncoated (C and D). In cases where detergent-TM domain interactions remain constant, conformational changes may also alter SDS loading stoichiometry (E and F). Interconversions among all types of hairpin-detergent complexes are possible. See text for further discussion.