Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:82929 |
| Date | 07 December 2023 |
| Creators | Schenkel, Mathias, Ravamehr-Lake, Dorna, Czerniak, Tomasz, Saenz, James P., Krainer, Georg, Schlierf, Michael, Deber, Charles M. |
| Publisher | Elsevier |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:article, info:eu-repo/semantics/article, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 1879-2642, 184078, 10.1016/j.bbamem.2022.184078, info:eu-repo/grantAgreement/European Commission/H2020 | MSCA-IF-EF-ST/841466//A Single-Molecule Technology for Resolving Chaperone Action in Neurodegenerative Diseases/MicroSPARK, info:eu-repo/grantAgreement/Natural Sciences and Engineering Research Council/Discovery Grants Program/RGPIN-2016-05577//Conformational transitions of membrane-spanning peptides/, info:eu-repo/grantAgreement/VolkswagenStiftung/Forschungsförderung/93090//Life |
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