ABCMdb

ABCC7 p.Gln207Asn

None has been submitted yet.

Publications

PMID: 17949679 [PubMed] Wehbi H et al: "Positional dependence of non-native polar mutations on folding of CFTR helical hairpins."

No. Sentence Comment

3 In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Login to comment
4 Over 95% of the backbone resonances of a 15 N,13 C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Login to comment
5 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
13 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence ⁎ Corresponding author. Login to comment
43 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
46 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
94 Here, the double mutant Q207N/V232E migrates significantly faster than wild type, indicating that the N/E pair is likely involved in side chain-side chain interactions analogously to the Q/D pair at the corresponding positions in the TM3/4-V232D single mutant [22]. Login to comment
95 In contrast, replacement of wild type Q237 in TM4 with the non-polar Leu residue (mutant Q237L) creates a slower migrating hairpin vs. wild type (Fig. 1a), suggesting that some pre-existing inter-helical interactions - conceivably attributable to a network of H-bonding interactions - have been removed. Login to comment
96 The helix-helix interactions influenced by polar mutants among the positions studied are summarized in Fig. 1b, where it is seen that constructs involving I231 and V232 with combinations of TM4 D-mutations, as well as various combinations of Q207N with D and E mutants in TM4, migrate between 7 and 20% faster than the WT construct. Login to comment
123 (a) Relative migration rates of wild type CFTR TM3/4 and mutants Q237L and Q207N-V232E. Login to comment
127 Note that the vertical axis is given as "% apparent MW decrease" such that the actual band appearance of all these mutants on gels is similar to Q207N-V232E in (A), i.e., faster migration than wild type. Login to comment
141 (a) WT; (b) V232D; (c) I231D; and (d) Q207N/V232E. Login to comment
142 Spectra were recorded in 115 mM PFO at 45 °C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
153 We then examined the double mutant Q207N/V232E-TM3/4 in which the potential polar partners are located in the sequence at the same positions as in V232D-TM3/4 (Q207 in TM3 and D232 in TM4). Login to comment
154 This mutant similarly migrates faster than V232D-TM3/4 (Fig. 1a, b). Login to comment
155 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
161 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
195 In contrast, the full 1 H-15 N HSQC spectra for V232D- and Q207N/V232E-TM3/4 exhibit striking similarities (not shown, but exemplified by the 'Gly box` comparison between Fig. 4 b and d), suggesting that these two mutants adopt similar conformations in the PFO micelle environment. Login to comment
196 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
200 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
202 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100° of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
208 Conclusion Features of the secondary structure of mutant V232D-TM3/4 helical hairpins of CFTR, along with comparisons to the WT and mutants I2231D and Q207N/V232E, have been determined using high resolution NMR spectroscopy. Login to comment
209 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment
6 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
14 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence Ìe; Corresponding author. Login to comment
44 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
47 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
97 The helix-helix interactions influenced by polar mutants among the positions studied are summarized in Fig. 1b, where it is seen that constructs involving I231 and V232 with combinations of TM4 D-mutations, as well as various combinations of Q207N with D and E mutants in TM4, migrate between 7 and 20% faster than the WT construct. Login to comment
124 (a) Relative migration rates of wild type CFTR TM3/4 and mutants Q237L and Q207N-V232E. Login to comment
128 Note that the vertical axis is given as "% apparent MW decrease" such that the actual band appearance of all these mutants on gels is similar to Q207N-V232E in (A), i.e., faster migration than wild type. Login to comment
156 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
162 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
201 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
203 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100&#b0; of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

No. Sentence Comment

121 A low amount (about 25%) of mature CFTR was observed only in mutant Q207N in the absence of VX-809. Login to comment
182 It was observed that in the absence of VX-809, the V510D mutation significantly improved the maturation of Q207L, Q207C, Q207E, Q207N and Q207S (Fig. 6A and B). Login to comment
183 Mature CFTR was the major product in Q207N/V510D (90% mature product) while mutants Q207L/V510D, Q207C/V510D, Q207E/V510D, and Q207S/V510D showed modest levels of mature CFTR (about 20-40% mature). Login to comment
283 Furthermore, Gln207 itself appeared to be important for maturation since all mutations to Gln207, including the conservative changes Q207N and Q207E (Fig. 2) inhibited maturation of the protein. Login to comment