Structural effects of extracellular loop mutations in CFTR helical hairpins.
Biochim Biophys Acta Biomembr
; 1860(5): 1092-1098, 2018 May.
Article
en En
| MEDLINE
| ID: mdl-29307731
ABSTRACT
Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Regulador de Conductancia de Transmembrana de Fibrosis Quística
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Mutación
Límite:
Humans
Idioma:
En
Revista:
Biochim Biophys Acta Biomembr
Año:
2018
Tipo del documento:
Article
País de afiliación:
Canadá