A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers

ABSTRACT We previously reported the equilibrium dimerization reaction of the CLC-ec1 Cl - /H + transporter in 2:1 POPE/POPG membranes (Chadda et al. 2016). This was determined by measuring the probability distributions of subunit capture into extruded liposomes by single-molecule photobleaching analysis across a wide range of subunit/lipid mole fraction densities. In this approach, knowledge of the liposome size distribution is necessary in order to correct the data for random co-capture events and extract the underlying dimerization reaction. For this we used a previously reported cryo-electron microscopy (cryo-EM) measured size distribution of 400 nm extruded liposomes made of E. coli polar lipids (Walden et al. 2007). While the model and data agreed at low densities, we observed systematic inaccuracies at higher densities limiting our ability to extract F Dimer in this range. To address this issue, we measured the 400 nm extruded 2:1 POPE/POPG liposome size distribution by cryo-EM and found that there is a small, but significant amount of larger liposomes in the population. Re-analysis of the I201W/I422W ‘WW’ photobleaching data using this distribution shows that the protein is monomeric in the membrane and can serve as an experimental control. Dimer controls were constructed by glutaraldehyde cross-linking of C85A/H234C ‘WT’ or introducing R230C/L249C, which forms a spontaneous disulfide bond. Determination of F Dimer based on the experimental controls yields improved fits and no change in the previously reported ΔG ° values, providing an alternate model-free approach to measuring CLC-ec1 dimerization in membranes.