Counting Membrane Proteins in Liposomes for Single-Molecule Microscopy

We can learn a lot by studying membrane proteins using single-molecule techniques. For example, electrical recordings of single ion channels have provided a wealth of information about key functional states. To obtain structural information, fluorescence microscopy can be used to monitor single-molecule Forster resonance energy transfer revealing dynamic conformational details. Experiments of this sort are being done but usually examine the protein in detergent micelles rather than a lipid environment. However, for many questions, studying the protein in lipid membranes is essential. While it is possible to carry out microscopy on proteins in supported membranes, formation of the bilayer can be complicated and often limits the lipid conditions. Another method is to simply examine the protein in liposomes, the benefits being that it is quick, easy and reconstitution is robust under different lipid compositions. In order to prepare this approach for single-molecule studies, we must establish the conditions in which we have a single protein molecule per liposome. Reconstitution statistics follows a Poisson distribution, which can be measured as a function of protein density and liposome size. Here we measure the size distributions of liposomes formed with E. Coli polar lipids and those containing mixtures of phosphatidyl-ethanolamine (PE), -glycerol (PG) and -choline (PC), by cryo-electron microscopy and fluorescence intensity of Alexa-488 labelled PE by total internal reflection fluorescence microscopy. The protein occupancy is measured as a function of protein density by photo-bleaching analysis of the Cl-/H+ transporter CLC-ec1 labelled with Cyanine-5. The results provide a foundation for future single-molecule studies of membrane proteins, such as the identification of conformational changes as well as the determination of protein stoichiometry with respect to studying the thermodynamics of protein association in membranes.