ABCC7 p.Glu402Cys
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PMID: 26149808
[PubMed]
Chong PA et al: "Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization."
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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