ABCMdb

ABCC7 p.Glu217Ser

None has been submitted yet.

Publications

PMID: 17516627 [PubMed] Wehbi H et al: "Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin."

No. Sentence Comment

69 We therefore hypothesized that the S222/E217 pair might form an intraloop interaction that stabilizes the native hairpin conformation and systematically replaced each side chain with Gly in the double mutant cycle consisting of S222G, E217G, S222G/E217G, and E217S/S222E. Login to comment
70 The unequal migration rates of the S222G and E217G hairpins in this cycle (6.8 ( 0.7 vs 12.0 ( 2.1, p ) 0.015), however, were inconsistent with an interaction between the two residues, and the double mutant E217S/S222E, where the positions of the hydrogen bond donor and acceptor are interchanged, exhibited the largest perturbation in hairpin compactness (percent increase of apparent molecular weight compared to wild type ) 26%) than any others encountered in this work. Login to comment
95 Thus, the mutants that migrated more slowly than TM3/4 WT on SDS-PAGE gels, among them Q220R and E217S, generally displayed increased helicity compared to TM3/4 WT (Table 1, Figure 4A). Login to comment
97 When the changes in TM3/4 WT hairpin migration were compared to changes in overall hairpin helicity, a strong correlation (R ) 0.79) was observed (Figure 5), leading us to propose that increases in non-native R-helix structure within ECL2 might Table 1: Migration Behavior on SDS-PAGE Gels of Single and Double Mutants in the Loop Region of CFTR TM3/4 Constructs % change in apparent MW on SDS-PAGE mutant vs TM3/4 WT in WT loop mutantsa vs TM3/4 V232D in V232D loop mutantsa Pb E217G 6.8 ( 0.7 E217S 11.1 ( 3.4 5.4 ( 1.4 0.056 Q220R 15.2 ( 1.1 Q220G 0.3 ( 0.4 Q220N 2.1 ( 1.3 0.5 ( 0.3 0.108 Q220K 14.1 ( 1.0 Q220W 13.1 ( 1.3 11.5 ( 0.9 0.157 Q220E -11.1 ( 1.1 -4.0 ( 0.3 <0.001 S222G 12.0 ( 2.1 1.1 ( 0.6 0.001 S222E -0.3 ( 2.4 1.3 ( 0.5 0.512 E217G/S222G 12.4 ( 1.9 E217S/S222E 26.1 ( 4.5 averagec 10.4 ( 7.3 4.0 ( 4.2 0.067 a Values are the percentage difference vs TM3/4 WT or TM3/4 V232D migration of SDS-PAGE gels. Login to comment
116 We found that the Q220E, S222G, and E217S replacements exhibited a relatively less pronounced effect on migration in the TM3/4 V232D hairpin than the TM3/4 WT background with high (p < 0.01 for Q220E and S222G) or detectable but marginal (p ) 0.056 for E217S) statistical significance. Login to comment
148 Moreover, in construct E217S/S222E, the net charge is not changed vs WT, yet the mutant runs at +26%. Login to comment

PMID: 19181854 [PubMed] Rath A et al: "Detergent binding explains anomalous SDS-PAGE migration of membrane proteins."

No. Sentence Comment

61 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to 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
97 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment
106 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
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
137 The E217S/S222E mutant is marked with an asterisk. Login to comment
138 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 Trendline P value in the absence of E217S/S222E is 0.002. Login to comment