Abstract
Estimating the number of subunits of a purified novel membrane protein is challenge. There are various methods for determining stoichiometry accurately in detergent, but often we are interested in the protein structure in the native solvent environment - the lipid bilayer. Here we describe a robust and widely applicable method to estimate the number of subunits in a purified membrane protein sample using single molecule fluorescent imaging. First, the multimeric protein complex is solubilized in detergent and purified. Second, it is quantitatively labelled with Cy5-maleimide and reconstituted into liposomes made of E. coli polar lipids or 2:1 POPE/POPG, doped with 0.3% of AF488-NHS ester labelled POPE. Following multiple freeze/thaw cycles to form multilamellar vesicles, the proteoliposomes are extruded through polycarbonate filters of 30, 100 and 400 nm pore diameter resulting in reproducibly distinct size distributions. Reconstitution follows the Poisson distribution resulting in liposomes containing either 0, 1, 2 or more protein molecules. The apparent protein occupancy into liposomes depends on protein:lipid density, efficiency of fluorescent labelling, liposome surface area and the subunit stoichiometry of the protein complex. Liposome size distributions are determined by cryo-electron microscopy whereas the protein density and fluorescent-labeling is controlled during the reconstitution step. We measure the first three terms in the Poisson distribution: F0 (unoccupied liposomes), F1 (single occupancy) and F2 (double occupancy) by single molecule imaging of the fluorescent proteoliposomes to measure protein/lipid co-localization and photobleaching of protein conjugated fluorophores. We test our model with a CLC-ec1 engineered monomeric construct, the native CLC-ec1 homodimer and tetrameric KcsA K+ channel. The methods outlined in this study can be used to determine the subunit stoichiometry of unknown purified membrane protein complexes in a variety of liposome environments.