We have used fluorescence microscopy, fluorescence photobleaching recovery (FPR), and atomic force microscopy (AFM) to investigate the formation of tethered lipid bilayers on plane aluminum oxide or glass surfaces. The bilayers were assembled with the help of a two-step methodology recently proposed for microporous templates (Proux-Delrouyre et al. J. Am. Chem. Soc. 2001, 123, 8313). The first step consists of the accumulation of intact biotinylated vesicles (PC + DOPE) on a streptavidin sublayer itself immobilized on the substrate. The second step, clearly time separated, is the deliberate triggering of bilayer formation with the help of poly(ethylene glycol) (PEG), a fusion agent of lipidic vesicles. AFM and FPR measurements confirm that the vesicles do not spontaneously fuse during the first step provided that the streptavidin sublayer is present on the substrate. On the contrary, the treatment with PEG provokes the fast formation of a continuous lipid bilayer, as attested at the hundred nanometer scale by the AFM images and at the hundred micrometer scale by the lateral diffusion of a fluorescent probe (D ) 2.2 × 10 -8 cm 2 s -1 for NBD-DMPE at 22 °C). † Part of the Langmuir special issue entitled The Biomolecular Interface.

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.