ABCC7 p.Glu402Cys
| Predicted by SNAP2: | A: N (53%), C: N (57%), D: N (97%), F: D (59%), G: D (59%), H: N (53%), I: N (78%), K: N (61%), L: D (53%), M: N (78%), N: N (57%), P: D (66%), Q: N (57%), R: D (66%), S: N (53%), T: N (53%), V: N (53%), W: D (63%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, F: D, G: N, H: N, I: N, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: D, |
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[hide] Deletion of Phenylalanine 508 in the First Nucleot... J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6. Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PMID:26149808]
Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.
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No. Sentence Comment
56 A single cysteine mutant of NBD1, E402C NBD1 èc;RIèc;RE, was generated on a Cys-less NBD1 (C491V, C524T, C590V, and C592V) background by site-directed mutagenesis and confirmed by DNA sequencing.
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ABCC7 p.Glu402Cys 26149808:56:34
status: NEW60 The tempo-maleimide spin label was conjugated to E402C, by first reducing the cysteine with 5 mM of freshly added DTT followed by extensive buffer exchange into a buffer with no DTT and subsequent incubation with a 5-fold excess of tempo-maleimide.
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ABCC7 p.Glu402Cys 26149808:60:49
status: NEW71 Paramagnetic relaxation enhancement experiments were measured using standard HSQC experiments on a sample containing 525 òe;M tempo-maleimide-labeled natural abundance E402C NBD1 èc;RIèc;RE and 175 òe;M 15 N-labeled WT NBD1 èc;RIèc;RE in the absence of reductant.
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ABCC7 p.Glu402Cys 26149808:71:172
status: NEW284 The single cysteine residue, E402C, was introduced on a Cys-less NBD1 èc;RIèc;RE (C491V, C524T, C590V, and C592V).
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ABCC7 p.Glu402Cys 26149808:284:29
status: NEW287 E402C NBD1 èc;RIèc;RE could be expressed and purified in the amounts required for NMR.
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ABCC7 p.Glu402Cys 26149808:287:0
status: NEW289 To ensure that E402C NBD1 èc;RIèc;RE is folded similarly to WT NBD1 èc;RIèc;RE, spectra of the mutant were recorded and compared with the WT (Fig. 9a).
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ABCC7 p.Glu402Cys 26149808:289:15
status: NEW295 c, overlay of spectra of 15 N WT NBD1 èc;RIèc;RE recorded in the presence of a 3-fold excess of isotopically natural abundance tempo-maleimide-labeled E402C NBD1 èc;RIèc;RE recorded before (red) and after (black) reduction of the spin label with TCEP.
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ABCC7 p.Glu402Cys 26149808:295:159
status: NEW296 d, ribbon diagram of an NBD1 dimer with 15 N WT NBD1 èc;RIèc;RE (teal) bound to E402C WT NBD1 èc;RIèc;RE (gray, not observable in this experiment).
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ABCC7 p.Glu402Cys 26149808:296:88
status: NEW303 F508del Increases Exchange and Reduces Dimerization SEPTEMBER 18, 2015ߦVOLUME 290ߦNUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 22873 E402C NBD1 èc;RIèc;RE.
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ABCC7 p.Glu402Cys 26149808:303:143
status: NEW307 To detect dimers, a sample containing 525 òe;M tempo-maleimide-labeled, isotopically natural abundance E402C NBD1 èc;RIèc;RE and 175 òe;M 15 N-labeled WT NBD1 èc;RIèc;RE was prepared.
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ABCC7 p.Glu402Cys 26149808:307:107
status: NEW319 Note that no broadening is expected or observed near Cys-402, because the tempo-maleimide-labeled E402C NBD1 èc;RIèc;RE is not 15 N-labeled and thus not visible in these spectra.
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ABCC7 p.Glu402Cys 26149808:319:98
status: NEW