ABCMdb

PMID: 19181854

Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM
Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.
Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30., 2009-02-10 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Val232Asp The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. Login to comment 24 ABCC7 p.Pro205Ser ABCC7 p.Val232Asp We find that gel shifts strongly correlate (R2 ϭ 0.8) with changes in the SDS-loading capacity of these miniature membrane proteins, indicating that altered detergent binding explains anomalous SDS-PAGE behavior. Our results reveal a distinction between two CF-phenotypic mutants studied: V232D binds significantly less SDS than the WT protein while P205S SDS binding is indistinguishable from WT, indicating that CFTR dysfunction may arise variously as a consequence of altered protein-lipid interactions or via altered intra-protein contacts. Login to comment 42 ABCC7 p.Pro205Ser ABCC7 p.Gln220Trp ABCC7 p.Val232Lys ABCC7 p.Glu217Val We noted that certain hairpins ran as more diffuse bands than others (e.g., P205S and Q220W vs. V232K and E217V, see Fig. 1B). Login to comment 45 ABCC7 p.Pro205Ala ABCC7 p.Ala204Leu Five mutants (A204L, P205A/ Table 1. Login to comment 61 ABCC7 p.Val232Asp ABCC7 p.Pro205Ala ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment 62 ABCC7 p.Pro205Ser ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Glu217Ser ABCC7 p.Gln220Trp ABCC7 p.Ser222Glu ABCC7 p.Val232Lys ABCC7 p.Glu217Val ABCC7 p.Glu217Phe ABCC7 p.Val232Ala ABCC7 p.Gly228Leu V232D, V232A, P205S, and Q220W) migrated as WT within statistical significance; 2 were faster (V232D and V232K); and 4 were slower (G228L, E217V, E217F, and E217S/S222E). Login to comment 70 ABCC7 p.Val232Asp ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu ABCC7 p.Glu217Phe The mutant hairpins ranged in loading levels from 3.4-10 g SDS/g, with V232D binding significantly fewer SDS molecules than WT, and the E217F and E217S/S222E mutants binding significantly more. Login to comment 71 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu ABCC7 p.Glu217Phe All hairpins load more detergent than do intact cytochrome b5, KcsA, and Glut1 [range 0.7-1.7 g SDS/g protein, (8-10)], and the E217F and E217S/S222E mutants are apparently the highest reported binders among the admittedly limited numbers of membrane proteins evaluated to date. Login to comment 85 ABCC7 p.Glu217Ser ABCC7 p.Gln220Trp ABCC7 p.Ser222Glu ABCC7 p.Val232Lys ABCC7 p.Glu217Val ABCC7 p.Ala204Leu ABCC7 p.Glu217Phe ABCC7 p.Val232Ala ABCC7 p.Gly228Leu Gel shifts, SDS binding, helicity, and column MW of TM3/4 hairpins Hairpin* Gel shift (dMW, %) Bound SDS (g/g) Helicity (MRE X 103)† Column MW (mut-wt, %)‡ V232Dcf -11 Ϯ 2.6 3.4 Ϯ 0.9 -17 Ϯ 1.2 ϩ19 Ϯ 1.5 V232K -10 Ϯ 3.0 3.8 Ϯ 0.6 -16 Ϯ 1.1 ϩ5.6 Ϯ 1.6 A204L -2.2 Ϯ 2.3 6.0 Ϯ 0.7 -18 Ϯ 1.3 ϩ3.4 Ϯ 1.6 P205A/V232Dcf ϩ0.12 Ϯ 5.2 4.7 Ϯ 0.4 -19 Ϯ 2.6 ϩ21 Ϯ 0.6 WT ϩ0.42 Ϯ 4.5 5.4 Ϯ 1.4 -18 Ϯ 2.2 0.0 Ϯ 0.79 V232A ϩ3.6 Ϯ 3.7 5.2 Ϯ 0.4 -18 Ϯ 0.9 ϩ6.1 Ϯ 1.9 P205Scf ϩ4.7 Ϯ 6.0 4.7 Ϯ 1.0 -18 Ϯ 0.7 ϩ4.4 Ϯ 1.6 Q220W ϩ6.3 Ϯ 2.4 5.0 Ϯ 0.7 -21 Ϯ 2.7 -4.9 Ϯ 1.3 G228L ϩ14 Ϯ 5.1 6.9 Ϯ 1.4 -23 Ϯ 1.7 ϩ14 Ϯ 2.6 E217V ϩ28 Ϯ 1.3 6.7 Ϯ 1.0 -25 Ϯ 1.7 ϩ32 Ϯ 4.0 E217F ϩ29 Ϯ 2.8 9.4 Ϯ 1.9 -28 Ϯ 2.6 ϩ17 Ϯ 1.1 E217S/S222E ϩ29 Ϯ 7.6 10 Ϯ 2.3 -25 Ϯ 2.8 ϩ35 Ϯ 0.9 Glycophorin§ - 3.4 Ϯ 0.6 -9.5 Ϯ 1.2 ϩ83 Ϯ 5.1 *Mutant hairpins are listed in order of increasing gel shift. Login to comment 97 ABCC7 p.Val232Asp ABCC7 p.Pro205Ala ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment 106 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu In the presence of the iso-hydropathic mutant E217S/S222E, correlations between each parameter and hydropathy range from R2 values of 0.371 (for bound SDS vs. hydropathy, Fig. 4) to 0.582 (for gel shift vs. hydropathy, Fig. S3). Login to comment 113 ABCC7 p.Val232Asp Conversely, even though it migrates faster than WT on SDS-PAGE, the V232D hairpin reports a radius approximately 20% larger than WT (Table 2). Login to comment 114 ABCC7 p.Gln220Trp Of the remaining hairpins, nine decreased in apparent compactness vs. WT (range from ϩ3% to ϩ35%, see Table 2), while Q220W appeared somewhat more compact than WT. Login to comment 123 ABCC7 p.Val232Asp ABCC7 p.Pro205Ala However, even if SDS/ protein stoichiometry (and by extension, gel shift) remains unchanged, increases in the conformational flexibility of non-coated regions may alter the hairpin`s hydrodynamic radius (compare Fig. 5 B-E)-a potential explanation for the as-WT gel shift but increased hydrodynamic radius relative to WT of the P205A/V232D mutant. Login to comment 127 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu ABCC7 p.Glu217Phe It is possible that the 9-10 g SDS/g stoichiometry of E217F and the iso-hydropathic E217S/S222E mutant are representative of detergent loading by a fully ''denatured`` helical membrane protein (such as the ''caterpillar`` structure in Fig. 5F). Login to comment 137 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu The E217S/S222E mutant is marked with an asterisk. Login to comment 138 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu Correlation coefficients (R2) of the best fit line in the absence of E217S/S222E (red) or with all mutants (black) are shown. Login to comment 139 ABCC7 p.Glu217Ser ABCC7 p.Ser222Glu Trendline P value in the absence of E217S/S222E is 0.002. Login to comment 141 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Pro205Ala For example, V232D and P205A/V232D display larger than WT hydrodynamic radii on SEC-HPLC (ϩ19% and ϩ21%, respectively)-even though each Asp-containing mutant migrates faster or as-WT on PAGE. Login to comment 153 ABCC7 p.Val232Asp Our observation that the V232D mutant binds less SDS than WT TM3/4 may therefore indicate that the dysfunction in the full-length CFTR molecule caused by this mutation arises from altered protein-lipid associations. Login to comment 155 ABCC7 p.Pro205Ser On the other hand, the as-WT stoichiometry of the P205S protein-SDS complex suggests that this lesion could disrupt CFTR folding and/or dynamics by directly altering protein-protein interactions while maintaining native protein-lipid contacts. Login to comment