P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett...

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.