ABCC7 p.Ser434Cys
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PMID: 17036051
[PubMed]
Mense M et al: "In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer."
No.
Sentence
Comment
67
The product band detected with antibody against the NH2-terminal half channel was of identical molecular mass to that identified with antibody against the COOH-terminal half channel, strongly sug- 250 150 100 75 434 459 462 549 605 1248 1336 1347 1374 1379 kDa A 5 µA 300 s (1-633) S549C and (634-1480) 9CS+S1248C Washout Washout Washout (1-633) and (634-1480) 9CS+D1336C (1-633) S434C and (634-1480) 9CS B C 549 549 no C no C no C 1248 1248 462 462 1336 1336 1347 1347 605 605 459 459 434 434 1374 1379 1379 0 20 40 60 80 100 0 2 4 6 8 Whole-oocyteconductance/expressionlevel Normalized expression level Resting conductance Stimulated conductance 1374 40 µM forskolin 40 µM forskolin 40 µM forskolin Figure 4 Expression (A, B) and function (A, C) of split CFTR channels containing introduced cysteines.
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ABCC7 p.Ser434Cys 17036051:67:385
status: NEW82 'NBD1` composite site, with A462C and S1347C, S459C and V1379C, and S434C and D1336C At the NBD1 composite site, we first examined crosslinking between positions homologous to those tested successfully at the NBD2 composite site.
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ABCC7 p.Ser434Cys 17036051:82:68
status: NEW86 Crosslinking was weaker, but still evident, 250 150 100 75 kDa - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + fsk Anti-R-domainAnti-N-terminus BMOE BMH Background S434C S459C A462C S549C S605C - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + S1248C D1336C S1347C A1374C V1379C 250 150 100 75 50 Figure 5 The absence of efficient crosslinking when no, or only one, engineered cysteine is present.
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ABCC7 p.Ser434Cys 17036051:86:224
status: NEW101 250 160 105 75 50 Anti-R-domainAnti-N-terminus kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC X-link CFTR 1-633 1 2 3 4 5 6 7 8 + + - 23ЊC - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC fsk BMOE BMH X-link CFTR 634-1480 9 10 11 12 13 14 + + - 15 16 23ЊC 0ЊC23ЊC 0ЊC23ЊC 0ЊC23ЊC 0ЊC23ЊC fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + X-link CFTR 1-633 1 2 3 4 5 6 7 fsk BMOE BMH X-link CFTR 634-1480 8 9 10 11 12 13 14 - - + - + + + - + + - +- - - - + -- + + 250 160 105 75 50 kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + X-link CFTR 1-633 1 2 3 4 5 6 7 fsk BMOE BMH X-link CFTR 634-1480 8 9 10 11 12 13 14 - - + - + + + - + + - +- - - - + -- + + kDa 250 150 100 75 50 A B C (1-633) A462C and (634-1480) 9CS+S1347C (1-633) S459C and (634-1480) 9CS+V1379C (1-633) S434C and (634-1480) 9CS+D1336C Figure 8 Crosslinking across the 'NBD1` composite site.
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ABCC7 p.Ser434Cys 17036051:101:883
status: NEW108 (C) CFTR half channels (1-633) S434C (left panel) and (634-1480) 9CSþ D1336C (right panel), as well as crosslinked product (arrow labeled X-link, both panels).
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ABCC7 p.Ser434Cys 17036051:108:31
status: NEW123 The six include three crosslinks across the NBD1 composite site (between the NBD1 head, containing the Walker motifs, and the NBD2 tail, containing the ABC signature sequence: C462-C1347, C459-C1379, and C434- C1336), one crosslink between central regions of NBD1 and NBD2 (C605-C1374), one crosslink between the NBD1-tail 250 150 100 75 50 kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC fsk BMOE BMH X-link CFTR 1-633 X-link CFTR 634-1480 Anti-R-domainAnti-N-terminus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (1-633) S549C and (634-1480) 9CS+A1374CB 250 150 100 75 kDa - + + - + - - - + fsk BMOE BMH - + + - + - - - + - + + - + - - - + - + + - + - - - + S459C/S1248C S549C/D1336C S549C/V1379C S605C/D1336C 250 150 50 Anti-R-domainAnti-N-terminus - + + - + - - - + S434C/A1374C A Two engineered cysteine control experiments Figure 9 Tests of crosslinking between NBD1 and NBD2 using other combinations of the target cysteines.
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ABCC7 p.Ser434Cys 17036051:123:847
status: NEW187 Primers for cysteine insertions S434C, S459C, A462C, S549C, S605C, S1248C, D1336C, S1347C, A1374C and V1379C are given in Table I.
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ABCC7 p.Ser434Cys 17036051:187:32
status: NEW199 For recording macroscopic currents of split CFTR channels in excised patches (Figure 10), oocytes were Table I Forward primers for site-directed mutagenesis PCR C76S 50 -GCCCTTCGGCGATcgTTTTTCTGGAG-30 C276S 50 -CTGTTAAGGCCTACTcCTGGGAAGAAGC-30 C832S 50 -CGAAGAAGACCTTAAGGAGTcCTTTTTTGATGATATGGAGAGC-30 EagI site 50 -GGTAAAATTAAGCACAGcGGccGAATTTCATTCTGTTCTC-30 HA epitope 50 -CGGGCCGCCATGtAcccatAcGACGttccgGAttAcgcaAGGTCGCCTCTGG-30 CFTR 16CS C590A/C592A 50 -GGAGATCTTCGAGAGCgCTGTCgCTAAACTGATGGC-30 CFTR 16CS C590F/C592F 50 -GGAGATCTTCGAGAGCTtTGTCTtTAAACTGATGGC-30 CFTR 16CS C590L/C592L 50 -GGAGATCTTCGAGAGCctTGTCctTAAACTGATGGC-30 CFTR 16CS C590T/C592T 50 -GGAGATCTTCGAGAGCaCTGTCaCTAAACTGATGGC-30 CFTR 16CS C590V/C592V 50 -GGAGATCTTCGAGAGCgtcGTCgtTAAACTGATGGC-30 S434C 50 -CCTCTTCTTCAGTAATTTCTgtCTaCTTGGTACTCCTGTC-30 S459C 50 -GTTGGCGGTTGCTGGATgCACTGGAGCAGGCAAG-3 A462C 50 -GCTGGATCCACTGGGtgcGGCAAGACTTCACTTC-30 L549C 50 -GGTGGAATCACACtatGcGGAGGTCAACGAGCACG-30 S605C 50 -GGATTTTGGTCACaTgTAAAATGGAAC-30 S1248C 50 -CCTCTTGGGAAGAACCGGtTgtGGGAAGAGTAC-30 D1336C 50 -GTTTCCTGGGAAGCTTtgCTTTGTCCTTGTGG-30 L1346C 50 -GGATGGGGGCTCTGTCTgtAGTCATGGCCACAAGC-30 A1374C 50 -GATGAACCAAGCtgTCATTTAGATCC-30 V1379C 50 -GCTCATTTAGATCCgtgcACATACCAAATAATTCG-30 The underlined bases are the codons for the introduced serines, cysteines or other residues; lowercase letters mark base changes from the original sequence, including those for introducing diagnostic restriction endonuclease sites.
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ABCC7 p.Ser434Cys 17036051:199:758
status: NEW
PMID: 8995353
[PubMed]
Loo TW et al: "Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators."
No.
Sentence
Comment
64
In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown).
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ABCC7 p.Ser434Cys 8995353:64:372
status: NEW