title: Design and characterization of a membrane protein unfolding platform in lipid bilayers.
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.
authors: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors: Vincent G
authors: VG
authors: Nadeau
authors: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors: Anqi
authors: A
authors: Gao
authors: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors: Charles M
authors: CM
authors: Deber
mesh_terms: D000595:Amino Acid Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding
chemical_list: C505032:CFTR protein, human; D008051:Lipid Bilayers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator