ArticlePloS one2015
Design and characterization of a membrane protein unfolding platform in lipid bilayers.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
What it found
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
3 citing papers in PubMed, 3 citations in OpenAlex.
- Steric trapping strategy for studying the folding of helical membrane proteins.Methods (San Diego, Calif.) · 2024Review
- Methods to study folding of alpha-helical membrane proteins in lipids.Open biology · 2022Review
- How physical forces drive the process of helical membrane protein folding.EMBO reports · 2022Review
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
Abstract
Accurate measurement of membrane protein stability--and particularly how it may vary as a result of disease-phenotypic mutations--ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding--results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.