ABCC7 p.Gly228Leu
| Predicted by SNAP2: | A: N (82%), C: D (63%), D: D (80%), E: D (85%), F: D (71%), H: D (85%), I: D (75%), K: D (91%), L: D (53%), M: D (80%), N: D (71%), P: D (66%), Q: D (80%), R: D (91%), S: N (57%), T: D (71%), V: D (71%), W: D (85%), Y: D (71%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, H: D, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
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[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).
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ABCC7 p.Gly228Leu 19181854:62:132
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.
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ABCC7 p.Gly228Leu 19181854:85:819
status: NEW