ABCMdb

ABCC7 p.Glu217Phe

ClinVar:	c.650A>G , p.Glu217Gly D , Pathogenic
CF databases:	c.650A>G , p.Glu217Gly (CFTR1) ? , The mutation was detected by heteroduplex analysis in a 2-year old male Polish patient with high sweat cloride (60-80 meq/l), pancreatic sufficiency, and moderate lung disease. His other CF mutation is unknown. It was also found by Yoshimura in 1999, in the CFTR alleles of a single patient with diffuse panbronchiolitis who has Q1352 H in the other allele.
Predicted by SNAP2:	A: N (66%), C: D (59%), D: N (93%), F: D (80%), G: N (66%), H: N (53%), I: D (59%), K: N (72%), L: D (59%), M: D (53%), N: N (82%), P: D (59%), Q: N (66%), R: D (66%), S: N (66%), T: N (66%), V: D (53%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: D, D: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] Detergent binding explains anomalous SDS-PAGE migr... Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30. 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 [PMID:19181854]

Abstract [show]
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.

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No. Sentence Comment
62 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 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 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 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
127 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

[hide] Sequence hydropathy dominates membrane protein res... Biochemistry. 2012 Aug 7;51(31):6228-37. Epub 2012 Jul 25. Nadeau VG, Rath A, Deber CM
Sequence hydropathy dominates membrane protein response to detergent solubilization.
Biochemistry. 2012 Aug 7;51(31):6228-37. Epub 2012 Jul 25., [PMID:22779403]

Abstract [show]
The ability to predict from amino acid sequence how membrane protein structures will respond to detergent solubilization would significantly facilitate experimental characterization of these molecules. Here we have investigated and compared the response to solubilization by the "mild" n-dodecyl-beta-d-maltoside (DDM) and "harsh" sodium dodecyl sulfate (SDS) of wild-type and point mutant "hairpin" (helix-loop-helix) membrane proteins derived from the third and fourth TM segments of the human cystic fibrosis transmembrane conductance regulator (CFTR) and the intervening extracellular loop. Circular dichroism spectroscopy, size-exclusion chromatography, and pyrene fluorescence spectroscopy were used to evaluate the secondary structures, hairpin-detergent complex excluded volumes, and hairpin compactness of the detergent-solubilized sequences. Sequence hydrophobicity is found to be the dominant factor dictating membrane protein response to detergent solubilization by DDM and SDS, with hairpin secondary structure exquisitely sensitive to mutation when DDM is used for solubilization. DDM and SDS differ principally in their ability to promote approach of TM segment ends, although hairpin compactness remains sensitive to point mutations. Our overall findings suggest that protein-protein and protein-detergent interactions are determined concomitantly, with the net hydropathy of residues exposed to detergent dominating the observed properties of the solubilized protein.

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89 We further noted that the spectra of mutant hairpins solubilized in DDM exhibited a gradation of intensities, whereas SDS-solubilized mutant spectra could be divided into "low helicity" (WT, V232D, A204L, and P205S) or "high helicity" (E217V, ES/SE, and E217F) clusters. Login to comment
96 The correlation of hydropathy levels in TM3/4 mutants and helicity in both detergents was also apparent in the rank ordering of mutants according to their molar residue ellipticity at 222 nm (Figure 2, right), with the exception of three mutants: E217F, E217V, and ES/SE (Figure 2A). Login to comment
189 We were thus excited to observe that solubilization by DDM supports a distance <10 Å between pyrene molecules conjugated at the ends of TM3 and TM4, giving rise to a subset of "compact" mutants (P205S, E217V, E217F, and A204L) (Figure 4B, left). Login to comment
193 Consistent with this possibility, the "high helicity" group of SDS-solubilized mutants (E217F, E217V, and ES/SE) has E:M ratios that do not exceed the negative control. Login to comment