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.
Highlights
Protein stability is the ultimate determinant of folding
We report the adaption of this method to the ‘hairpin’ system [18,19,20] in lipid bilayers, in which the hairpin consists of an 87-residue helix-loop-helix construct derived from transmembrane helices 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR)
Our results indicate that the steric trap is amenable to the TM3/4 hairpin system, while highlighting the variables that arise in the stability analysis of these constructs reconstituted in lipid bilayers
Summary
Protein stability is the ultimate determinant of folding. Protein stability can be quantified as a reversible change from a folded state to a fully unfolded state [1]— an energy difference that can vary significantly upon mutation of the protein sequence in a given environment. Membrane Protein Unfolding in Bilayers treated with increasing levels of a denaturant, which could consist of chaotropic agents (urea, guanidinium chloride), harsh detergents (SDS), temperature (cold or heat), or external mechanical force. Unfolding can be monitored through structural or functional loss until the protein has reached its unfolded state. This unfolding must be reversible and return the protein to its initial folded state upon removal of the denaturant
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