ABCC7 p.Glu115Ser
| Predicted by SNAP2: | A: N (93%), C: N (87%), D: N (93%), F: N (82%), G: N (87%), H: N (93%), I: N (87%), K: N (93%), L: N (87%), M: N (87%), N: N (93%), P: N (93%), Q: N (97%), R: N (82%), S: N (97%), T: N (97%), V: N (93%), W: D (53%), Y: N (93%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Mapping of cystic fibrosis transmembrane conductan... J Biol Chem. 1994 Jul 15;269(28):18572-5. Chang XB, Hou YX, Jensen TJ, Riordan JR
Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.
J Biol Chem. 1994 Jul 15;269(28):18572-5., [PMID:7518437]
Abstract [show]
Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function. However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer. Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins.
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23 1B).A PCR fragment coding for the E115S change was introduced into EL0 to generate a consensus glycosylation site on extracellular loop 1 (ELI, Fig.1B).Fourdifferent PCR fragmentscontaininginsertions coding for peptides NTS, GSNNSH, HRNQS, and NTS were inserted into EL0 at extracytoplasmic loops 2, 3, 5, and 6, respectively, to generate potential glycosylation sites on each putative extracellular loop (Fig. 1B).
X
ABCC7 p.Glu115Ser 7518437:23:34
status: NEW64 The extent of sequence perturbation employed to insert these sites rangedfrom a single aminoacid change (E115S)in EL1 to the insertionof 6 additional residues inEL3.
X
ABCC7 p.Glu115Ser 7518437:64:105
status: NEW