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- W1994543256 abstract "Experimental determination of the free energy of membrane protein oligomerization in lipid bilayers is difficult. There are few membrane protein systems that allow for this equilibrium measurement, and for strong complexes, the amount of dilution necessary to observe dissociation often obscures experimental detection. We have devised a new model system for measuring equilibrium dimerization in membranes, using the homodimeric CLC-ec1 Cl-/H+ antiporter. A mutated version of CLC-ec1 that bears a single tryptophan substitution on the dimerization interface (CLC-W) shifts the protein to the monomeric state in detergent micelles. We quantitatively labelled CLC-W with Cy3 or Cy5 fluorophore and reconstituted the protein into 2:1 POPE/POPG lipids. Then, we measured macroscopic Forster resonance energy transfer (FRET) in large membranes to assess CLC-W dimerization. Mixing CLC-W-Cy3 and CLC-W-Cy5 in detergent before reconstitution, or fusing CLC-W-Cy3 liposomes with CLC-W-Cy5 liposomes yields a FRET signal indicative of dimer formation and equilibrium exchange. We also confirmed that the protein was folded, by measuring Cl- transport function. We then investigated dissociation of CLC-W by “traditional” dilution in membranes. Since wild-type CLC-ec1 does not undergo dimer exchange, we used CLC-ec1-Cy3 mixed with CLC-ec1-Cy5 to determine the “all-monomer” background signal, and co-labelled CLC-ec1-Cy3/Cy5 to determine the “all-dimer” FRET signal, at various Cy3:Cy5 labeling ratios and protein:lipid densities. With this, we observe that CLC-W begins to dissociate at a density of 1 protein per 300,000 lipids. At densities below 1 protein per 650,000 lipids, macroscopic FRET is obscured by background scattering and so we turn to single-molecule fluorescence microscopy to measure CLC-W stoichiometry by fluorophore photo-bleaching. These studies demonstrate that we have pushed the observable limit of this reaction, expanding our ability to measure the free energy of membrane protein assembly in lipid bilayers." @default.
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- W1994543256 date "2015-01-01" @default.
- W1994543256 modified "2023-09-28" @default.
- W1994543256 title "Determining the Free Energy of Membrane Protein Dimerization in Lipid Bilayers" @default.
- W1994543256 doi "https://doi.org/10.1016/j.bpj.2014.11.241" @default.
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