ABCMdb

PMID: 17949679

Wehbi H, Gasmi-Seabrook G, Choi MY, Deber CM
Positional dependence of non-native polar mutations on folding of CFTR helical hairpins.
Biochim Biophys Acta. 2008 Jan;1778(1):79-87. Epub 2007 Sep 15., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Val232Glu 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 6 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Val232Glu 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 14 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Val232Glu 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 35 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment 36 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment 43 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 44 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 47 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 52 ABCC7 p.Val232Asp Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 μg/mL ampicillin. Login to comment 53 ABCC7 p.Val232Asp Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 bc;g/mL ampicillin. Login to comment 73 ABCC7 p.Val232Asp Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 °C. Login to comment 74 ABCC7 p.Val232Asp Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 &#b0;C. Login to comment 94 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Gln237Leu 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 ABCC7 p.Gln207Asn ABCC7 p.Gln237Leu 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 97 ABCC7 p.Gln207Asn 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 99 ABCC7 p.Val232Asp ABCC7 p.Val232Asp Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 °C is shown in Fig. 2. Login to comment 100 ABCC7 p.Val232Asp ABCC7 p.Val232Asp Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 &#b0;C is shown in Fig. 2. Login to comment 104 ABCC7 p.Val232Asp Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment 105 ABCC7 p.Val232Asp Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment 107 ABCC7 p.Val232Asp Hα, Cα, Cβ, CO, and NH chemical shifts of V232D-TM3/4 residues were analyzed using TALOS [51]. Login to comment 108 ABCC7 p.Val232Asp ABCC7 p.Val232Asp The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment 109 ABCC7 p.Val232Asp The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment 118 ABCC7 p.Val232Asp 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment 119 ABCC7 p.Val232Asp 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment 123 ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Gln237Leu (a) Relative migration rates of wild type CFTR TM3/4 and mutants Q237L and Q207N-V232E. Login to comment 124 ABCC7 p.Gln207Asn ABCC7 p.Val232Glu ABCC7 p.Gln237Leu (a) Relative migration rates of wild type CFTR TM3/4 and mutants Q237L and Q207N-V232E. Login to comment 127 ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 128 ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 129 ABCC7 p.Val232Asp However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment 130 ABCC7 p.Val232Asp However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment 131 ABCC7 p.Val232Asp Analysis of 1 H-15 N HSQC spectra of WT-TM3/4 and its various mutants in PFO micelles To probe for global conformational changes of TM3/4 upon introducing a polar residue in TM4, the 1 H-15 N HSQC spectrum of wild type CFTR TM3/4 was acquired in PFO micelles under the same buffer conditions as V232D-TM3/4. Login to comment 132 ABCC7 p.Val232Asp ABCC7 p.Val232Asp With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment 133 ABCC7 p.Val232Asp With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment 134 ABCC7 p.Val232Asp Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment 135 ABCC7 p.Val232Asp Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment 141 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu (a) WT; (b) V232D; (c) I231D; and (d) Q207N/V232E. Login to comment 142 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 143 ABCC7 p.Val232Asp Spectra were recorded in 115 mM PFO at 45 &#b0;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 144 ABCC7 p.Val232Asp As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment 145 ABCC7 p.Val232Asp As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment 146 ABCC7 p.Val232Asp However, among the four spectra shown, significant chemical shift changes are observed for several of G213, G226, G228, G239 and G241 resonances between WT and V232D-TM3/4. Login to comment 147 ABCC7 p.Val232Asp ABCC7 p.Val232Asp As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment 148 ABCC7 p.Val232Asp As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment 149 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp In order to further investigate the conformational changes of TM3/4 upon introduction of a polar residue into TM4, we next performed a comparative analysis of amide 1 H/15 N chemical shifts in the 1 H-15 N HSQC spectra of WT-TM3/4 vs. I231D-TM3/4 which contains a polar mutation at the position preceding the CF-phenotypic mutation V232D-TM3/4. Login to comment 150 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp Note that this mutant exhibited a faster migration rate than both WT-TM3/4 and V232D-TM3/4 on SDS-PAGE [22] (Fig. 1b). Login to comment 151 ABCC7 p.Val232Asp ABCC7 p.Val232Asp From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment 152 ABCC7 p.Val232Asp From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment 153 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu This mutant similarly migrates faster than V232D-TM3/4 (Fig. 1a, b). Login to comment 155 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 156 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 160 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu While some mutants do contain a change in charge vs. WT, previous work from our laboratory has established that CFTR TM3/4 migration patterns are not simple functions of charge: (i) WT TM3/4 and the double mutant Q207L/V232D the same migration rates while V232D migrates significantly faster than WT [20]; (ii) Asp substitutions at 20 different positions along TM4 between residues 221 and 241 produce TM3/4 hairpins that migrate 3-12% faster than WT [22]; if introduction of a single negative charge was the dominating effect, all 20 mutants should display similar migration rates. Login to comment 161 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 162 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 174 ABCC7 p.Val232Asp We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment 175 ABCC7 p.Val232Asp We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment 187 ABCC7 p.Val232Asp Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment 188 ABCC7 p.Val232Asp Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment 189 ABCC7 p.Val232Asp In addition to these "Gly box" effects, chemical shift changes vs. WT in the amide proton dimension of virtually all of the residues in the V232D-TM3/4 spectrum, including A223, A225, and G226 from the turn, as well as W216, L218, L219, Q220, and A221 near the C-terminus of the TM3 helix (data not shown), confirming that the effect is not limited to immediate neighbors. Login to comment 190 ABCC7 p.Val232Asp ABCC7 p.Val232Asp These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment 191 ABCC7 p.Val232Asp These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment 192 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment 193 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment 194 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp The full amide chemical shift analysis of the 1 H-15 N HSQC shows that similarly to the V232D mutant, all TM4 amides - along with residues from W216 through A221 (C-terminus of TM3) and from S222 to G226 (turn) - are highly affected by the I231D mutation (not shown), suggesting that this mutation introduces significant changes in packing relative to WT protein. Login to comment 195 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 197 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp 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 199 ABCC7 p.Ile231Asp Molecular dynamics simulations of WT TM3/4 vs. I231D-TM3/4 further support the view that a folded two-helix hairpin forms in a micellar environment, and that a side chain-side chain Q207-D231 H-bond provides additional stabilization for the folded state [64]. Login to comment 200 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 201 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 203 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 208 ABCC7 p.Val232Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Gln207Asn ABCC7 p.Val232Glu 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 210 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp 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