title: Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.
abstract: Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F-ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl-/H+antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known.
authors: The University of Iowa, Iowa City, IA, United States.
authors: R
authors: R
authors: Chadda
authors: The University of Iowa, Iowa City, IA, United States. Electronic address: janice-robertson@uiowa.edu.
authors: J L
authors: JL
authors: Robertson
mesh_terms: D019281:Dimerization; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007473:Ion Channels / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008245:Lysophospholipase / Q000737:chemistry / Q000302:isolation & purification*; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D055503:Protein Multimerization; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D006023:Glycoproteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008245:Lysophospholipase; C011711:lysolecithin acylhydrolase
keywords: Dimerization; Equilibrium; Lipid bilayers; Membrane protein; Photobleaching; Poisson; Single-molecule microscopy