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Detergent Binding Explains Anomalous SDS-PAGE Migration of Membrane Proteins

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Detergent Binding Explains Anomalous SDS-PAGE Migration of Membrane Proteins Detergent binding explains anomalous SDS-PAGE migration of membrane proteins Arianna Ratha,b, Mira Glibowickaa, Vincent G. Nadeaua,b, Gong Chena,b, and Charles M. Debera,b,1 aDivision of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and bDepartment of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Communicated by Donald M. Engelman, Yale University, New Haven, CT, December 23, 2008 (received for review December 19, 2007) Migration on sodium dodecyl sulfate-polyacrylamide gel electro- mon to globular polypeptides orient their hydrophobic faces toward phoresis (SDS-PAGE) that does not correlate with formula molecular the detergent tails and hydrophilic portions toward the micelle weights, termed ‘‘gel shifting,’’ appears to be common for membrane surface [reviewed in (1)]. Membrane proteins in some cases load proteins but has yet to be conclusively explained. In the present work, 2-fold greater amounts of SDS than globular polypeptides; exam- we investigate the anomalous gel mobility of helical membrane ples include the 3.4 g SDS/g stoichiometry of human erythrocyte proteins using a library of wild-type and mutant helix-loop-helix membrane glycoprotein glycophorin (6) and 4.5 g SDS/g bound to (‘‘hairpin’’) sequences derived from transmembrane segments 3 and CP-B2, a membrane protein from R. rubrum chromatophores (7). 4 of the human cystic fibrosis transmembrane conductance regulator Large SDS/protein aggregate stoichiometry is nevertheless not (CFTR), including disease-phenotypic residue substitutions. We find universal among membrane-soluble polypeptides: intact cyto- that these hairpins migrate at rates of ؊10% to ؉30% vs. their actual chrome b5 binds 1.2 g SDS/g protein (8); the KcsA potassium formula weights on SDS-PAGE and load detergent at ratios ranging channel tetramer 0.7 g SDS/g (9); and the human red cell glucose from 3.4–10 g SDS/g protein. We additionally demonstrate that transporter Glut1 1.7 g SDS/g (10). mutant gel shifts strongly correlate with changes in hairpin SDS Protein tertiary structure may affect both detergent-loading levels and polypeptide-SDS-PAGE migration rates. Disulfide ,(0.9 ؍ and with hairpin helicity (R2 ,(0.8 ؍ loading capacity (R2 indicating that gel shift behavior originates in altered detergent bonds, for example, reduce SDS binding to globular proteins by up binding. In some cases, this differential solvation by SDS may result to 2-fold (11), and have been linked to the anomalously fast from replacing protein-detergent contacts with protein-protein con- migration of unreduced vs. reduced lysozyme, presumably because tacts, implying that detergent binding and folding are intimately the intact disulfide bonds impose a more compact shape on the linked. The CF-phenotypic V232D mutant included in our library may enzyme (12). Similarly, in a previous study with helix-loop-helix or thus disrupt CFTR function via altered protein-lipid interactions. The ‘‘hairpin’’ model membrane proteins based on portions of the TM domain of the human cystic fibrosis transmembrane conductance observed interdependence between hairpin migration, SDS aggre- regulator (CFTR), we noted that increased SDS-PAGE migration gation number, and conformation additionally suggests that deter- rates were traceable to a compact S-S bridged conformation (13). gent binding may provide a rapid and economical screen for identi- The E. coli ␤-barrel membrane protein OmpA also binds less SDS fying membrane proteins with robust tertiary and/or quaternary as a folded than fully denatured species (14, 15), and moves through structures. gels according to structural compactness, with the folded form migrating faster than the completely denatured protein (16). ͉ ͉ ͉ ͉ detergent binding gel shifting membrane proteins SDS-PAGE While these overall observations suggest that there may be a sodium dodecyl sulfate specific relationship between protein structure and SDS loading, a unifying source for atypical membrane protein SDS-PAGE migra- etermination of protein molecular weight (MW) via polyacryl- tion has not emerged, and questions as to whether this detergent Damide gel electrophoresis (PAGE) in the presence of sodium ‘‘denatures’’ membrane proteins and/or their oligomeric forms are dodecyl sulfate (SDS) is a universally used method in biomedical often posed (17). In the present work, we establish a quantitative research. The technique requires that the proteins under investi- relationship between gel migration behavior and SDS aggregate gation on the gel and the proteins used for MW calibration adopt stoichiometry using a library of helical hairpins derived from equivalent shapes after SDS treatment. This condition is achieved CFTR. We find that gel shifts strongly correlate (R2 ϭ 0.8) with not by the complete unfolding of the proteins but rather by the changes in the SDS-loading capacity of these miniature membrane aggregation of SDS molecules at hydrophobic protein sites to proteins, indicating that altered detergent binding explains anom- induce ‘‘reconstructive denaturation,’’ where proteins adopt a con- alous SDS-PAGE behavior. Our results reveal a distinction be- formational mixture of ␣-helix and random coil [reviewed in (1)]. tween two CF-phenotypic mutants studied: V232D binds signifi- The resultant protein-detergent complexes consist of helical SDS- cantly less SDS than the WT protein while P205S SDS binding is coated polypeptide regions spatially separated by flexible and indistinguishable from WT, indicating that CFTR dysfunction may uncoated linkers, termed ‘‘necklace and bead’’ structures (2). arise variously as a consequence of altered protein-lipid interactions Individual sizes of the micellar ‘‘beads’’ can vary and seem to be or via altered intra-protein contacts. determined by amino acid sequence (3). A further proviso for MW Results estimation by SDS-PAGE is a consistent amount of detergent binding among proteins (4). Maximum SDS-binding levels are SDS-PAGE Mobility of Hairpins vs. Full-length Helical Membrane generally quoted at 1.4 g SDS/g protein, following the work of Proteins. To gain an understanding of the commonality and mag- Reynolds and Tanford that identified this weight ratio as nearly nitude of anomalous SDS-PAGE migration behavior observed in invariant for a variety of globular and erythrocyte ghost proteins (4). Subsequent investigations of SDS loading have refined this Author contributions: A.R. and C.M.D. designed research; A.R., M.G., and V.G.N. performed saturation value for globular proteins to as much as 1.5–2 g research; M.G. and G.C. contributed new reagents/analytic tools; A.R., V.G.N., and C.M.D. detergent/g protein (5). analyzed data; and A.R. and C.M.D. wrote the paper. Peptide segments that contain an abundance of hydrophobic The authors declare no conflict of interest. residues—such as the transmembrane (TM) sequences of mem- 1To whom correspondence should be addressed. E-mail: [email protected]. brane proteins—have helical regions expected to be embedded This article contains supporting information online at www.pnas.org/cgi/content/full/ within the SDS micelle interior, while amphipathic segments com- 0813167106/DCSupplemental. 1760–1765 ͉ PNAS ͉ February 10, 2009 ͉ vol. 106 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813167106 Downloaded by guest on September 28, 2021 Table 1. Gel shifts of various helical membrane proteins on SDS-PAGE MW (kDa) Gel shift dMW, Protein* App. Formula %† Ref. I. tartaricus F-type ATPase c subunit 41 Undecamer 53 97 Ϫ46 Monomer 6.5 8.8 Ϫ26 Archaeal ammonium transporter Amt-1 42 Trimer 90 134 Ϫ33 Monomer 33 45 Ϫ27 E. coli lactose permease 33 47 Ϫ30 43 Vitamin B12 transporter BtuC 26 35 Ϫ26 44 Hϩ/ClϪ exchange transporter ClC 38 51 Ϫ25 45 Potassium channel KcsA tetramer 60 76 Ϫ21 46 Phospholamban 47 Pentamer 29 30 Ϫ17 Monomer 9 6.1 ϩ48 Particulate methane monooxygenase 48 pmoA subunit 24 28 Ϫ14 pmoB subunit 47 46 2 pmoC subunit 22 30 Ϫ27 Glycerol facilitator channel GlpF 29 34 Ϫ15 49 E. coli MscS channel 50 Dimer 52 62 Ϫ16 Monomer 27 31 Ϫ12 Multidrug transporter AcrB 100 115 Ϫ13 51 Glycerol-3-phosphate transporter GlpT 45 50 Ϫ10 52 Spinach aquaporin 31 32 Ϫ353 Rhomboid family protease GlpG 31 31 0 54 M. tuberculosis MscL channel monomer 20 16 ϩ26 55 ␤2-adrenergic receptor 62 47 ϩ30 56 M. thermoautotrophicum MthK tetramer 200 149 ϩ34 57 *Proteins were selected from a list of non-redundant helical membrane proteins based on the availability of published SDS-PAGE data and are arranged in approximate order of faster-to-slower than expected migration BIOCHEMISTRY rates. The helical nature of each protein has been confirmed in a high-resolution structure. Oligomeric data were obtained only from gels where extrinsic cross-linking agents were absent. †The migration distances of proteins on SDS-PAGE gels reported in the papers examined, compared to standards on these gels. dMW ϭ (apparent MW Ϫ formula MW)/formula MW ϫ 100%. Negative dMWs indicate movement faster (at a lower apparent MW) than expected and vice-versa. full-length polypeptides, we surveyed electrophoretic migration others (e.g., P205S and Q220W vs. V232K and E217V, see Fig. 1B). data obtained from the literature for a non-redundant dataset of Spreading of bands on SDS-PAGE can reflect heterogeneity in helical membrane proteins. The apparent MWs among this group protein species or in protein-detergent complexes; given that each deviate widely from formula MW (Table 1) with gel shifts (i.e., sample loaded is fully purified, it is possible that the latter expla- migration on PAGE that does not correspond to formula MW) nation may lead to the comet-like appearance of certain bands. ranging from Ϫ46% (migration faster than formula MW) observed Hairpin apparent MWs were determined with reference to globular for the 11-mer of the F-type ATPase c subunit to ϩ48% (slower protein standards and normalized by formula MWs to obtain gel than formula MW) displayed by the phospholamban monomer.
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