ABCC7 p.His1402Ser
| Predicted by SNAP2: | A: D (91%), C: D (85%), D: D (91%), E: D (91%), F: D (91%), G: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (75%), P: D (95%), Q: D (80%), R: D (91%), S: D (85%), T: D (85%), V: D (91%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Thermal stability of purified and reconstituted CF... Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15. Aleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR
Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.
Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15., [PMID:26384709]
Abstract [show]
CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, DeltaF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied DeltaF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 degrees C and remain monodisperse at 4 degrees C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.
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No. Sentence Comment
112 When the H1402S mutations was added to the combined NBD1 modifications, the amount of CFTR expressed by the cells (and present in isolated membranes) was increased 3 to 5-fold compared to the wild-type and the mature/immature band intensity ratio as an index of maturation also was elevated (not shown).
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ABCC7 p.His1402Ser 26384709:112:9
status: NEW114 The thermal stability of the DRI/2PT/H1402S channel in isolated microsomal membranes was evaluated and it was found to retain full activity at 45 &#b0;C for at least 1 h (Fig. 3, top tracing) and remain active during a temperature ramp from 45 &#b0;C to 50 &#b0;C albeit with open state subconductances most evident in the extended lower tracing at 50 &#b0;C. Thus, the increased amounts of protein expressed and greater thermal stability encouraged us to proceed with purification and characterization of this variant.
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ABCC7 p.His1402Ser 26384709:114:37
status: NEW118 This number of cells yielded 1 g of microsomal membranes from which 2 mg of wild-type and 10 mg of the DRI/2PT/H1402S variant could be purified.
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ABCC7 p.His1402Ser 26384709:118:114
status: NEW120 The purified DRI/2PT/ H1402S protein was highly homogeneous as judged by heavily loaded SDS-PAGE gels (Fig. 4a) and appeared monodisperse in the low concentration of the DMNG (at 3 CMC) detergent in which it was purified (Fig. 4b).
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ABCC7 p.His1402Ser 26384709:120:22
status: NEW123 The gel filtration profile of the purified DRI/2PT/H1402S protein was unaltered after storage at 4 &#b0;C for at least two weeks and little changed when kept at 16 &#b0;C for this period of time (data not shown).
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ABCC7 p.His1402Ser 26384709:123:51
status: NEW128 In marked contrast to the minimal CFTR channel activity observed with the purified wild-type protein, the purified DRI/2PT/H1402S protein was fully active at temperatures of 25, 30 and 37 &#b0;C with very high open probability in all cases (Fig. 6, left 3 tracings).
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ABCC7 p.His1402Ser 26384709:128:123
status: NEW163 When this set of NBD1 stabilizing mutations were combined with the H1402S substitution in NBD2 the level of mature protein expression in BHK cells increased several fold, enabling purification of milligram quantities of homogeneous protein that remained monodisperse at concentrations >3 mg/ml in a low concentration of DMNG (3 CMC).
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ABCC7 p.His1402Ser 26384709:163:67
status: NEW171 Thermal sensitivity of NBD1 and NDB2 modified variant, DRI/2PT/H1402S.
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ABCC7 p.His1402Ser 26384709:171:63
status: NEW176 Purified DRI/2PT/H1402S CFTR protein before and after reconstitution.
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ABCC7 p.His1402Ser 26384709:176:17
status: NEW187 Thermal stability of purified and reconstituted DRI/2PT/H1402S CFTR.
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ABCC7 p.His1402Ser 26384709:187:56
status: NEW