P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer.
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.
Diociaiuti
Marco
M
Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. marco.diociaiuti@iss.it
Molinari
Agnese
A
Ruspantini
Irene
I
Gaudiano
Maria Cristina
MC
Ippoliti
Rodolfo
R
Lendaro
Eugenio
E
Bordi
Federico
F
Chistolini
Pietro
P
Arancia
Giuseppe
G
D015060:1,2-Dipalmitoylphosphatidylcholine; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D000251:Adenosine Triphosphatases / Q000737:chemistry; D002244:Carbon; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry; D007150:Immunohistochemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D011487:Protein Conformation
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000939:Epitopes; D008051:Lipid Bilayers; D008081:Liposomes; D015060:1,2-Dipalmitoylphosphatidylcholine; D002244:Carbon; D000251:Adenosine Triphosphatases