ABCC7 p.Glu217Val
| 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
42 We noted that certain hairpins ran as more diffuse bands than others (e.g., P205S and Q220W vs. V232K and E217V, see Fig. 1B).
X
ABCC7 p.Glu217Val 19181854:42:106
status: NEW62 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).
X
ABCC7 p.Glu217Val 19181854:62:139
status: NEW85 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.
X
ABCC7 p.Glu217Val 19181854:85:901
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
87 The intensity of mean residue ellipticity at 222 nm, for example, ranged by ~20 000 deg cm2 dmol-1 among the hairpin sequences (compare E217V and V232D, Figure 2A, left), a result that might not initially be anticipated for a "mild" detergent.
X
ABCC7 p.Glu217Val 22779403:87:136
status: NEW89 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.
X
ABCC7 p.Glu217Val 22779403:89:236
status: NEW96 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).
X
ABCC7 p.Glu217Val 22779403:96:254
status: NEW144 In DDM (Figure 4B, left), we found that the P205S (E:M = 0.87 ± 0.20), E217V (E:M = 0.78 ± 0.06), E271F (E:M = 0.76 ± 0.12) and A204L spectra (E:M = 0.69 ± 0.02) each displayed excimer fluorescence, and the E:M ratio of each of these "compact" mutants was statistically distinct from the negative control in DDM (E:M = 0.52 ± 0.02, 4.7 × 10-6 ≤ p ≤ 0.008).
X
ABCC7 p.Glu217Val 22779403:144:76
status: NEW146 In contrast, after SDS solubilization (Figure 4B, right), excimer emission above negative control (E:M = 0.21 ± 0.004) levels was present only in the A204L mutant (E:M = 0.32 ± 0.06, p = 0.027); the E217V mutant had an E:M ratio higher than the negative control, but this difference was of marginal significance (E:M = 0.28 ± 0.04, p = 0.062).
X
ABCC7 p.Glu217Val 22779403:146:209
status: NEW169 A further observation from the sets of CD curves shown in Figure 2 is that the spectra in SDS display ellipticity notably skewed toward the 208 nm lobe, while corresponding spectra in DDM generally have approximately equal ellipticity for the 208 and 222 nm lobes (exception: the loop-based E217V mutant).
X
ABCC7 p.Glu217Val 22779403:169:291
status: NEW189 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).
X
ABCC7 p.Glu217Val 22779403:189:207
status: NEW193 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.
X
ABCC7 p.Glu217Val 22779403:193:95
status: NEW