Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane.
A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.
Elie-Caille
Céline
C
CNRS UMR 6022, Université de Technologie de Compiègne, B.P. 20529, 60205 Compiègne, France.
Fliniaux
Ophélie
O
Pantigny
Jacques
J
Mazière
Jean-Claude
JC
Bourdillon
Christian
C
D000327:Adsorption; D045744:Cell Line, Tumor; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008561:Membrane Fusion; D008930:Mitochondria, Liver / Q000737:chemistry; D051336:Mitochondrial Membranes / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry*; D019809:Streptavidin; D013499:Surface Properties
D008051:Lipid Bilayers; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D019809:Streptavidin