Self-Assembly of Solid-Supported Membranes Using a Triggered Fusion of Phospholipid-Enriched Proteoliposomes Prepared from the Inner Mitochondrial Membrane¹

Abstract

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.

Native E. coli inner membrane incorporation in solid-supported lipid bilayer membranes

“…Our approach is similar to the method first used by Schneider et al in 1980, who investigated electron transfer mechanisms, 36 in which phospholipid vesicles were generated through sonication, mixed with the inner membrane of mitochondria; fusion was achieved through careful adjustments of the pH. This method was recently adapted by Elie-Caille et al, 37 who were also using a mitochondrial inner membrane in order to study the electron transfer machinery on solid-supported lipid membranes. In this case the sonicated phospholipid vesicles were partially biotinylated and, after mixing with the mitochondrial membranes, the two were sonicated together to promote fusion.…”

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity

“…A variety of methods have been developed to deposit the upper BLM leaflet on the tethering layer and incorporate a variety of ionophores, peptides, and transmembrane proteins. In one commonly used approach, the purified protein is reconstituted into liposomes, and the resulting proteoliposomes simultaneously deposit the upper BLM leaflet and the protein [26][27][28]. In a second method, the liposomes are used to deposit the upper BLM leaflet.…”

“…Previous attempts to form sBLM using microsomes containing membrane proteins did not yield an insulating bilayer membrane [28] and were not suitable to study the ion-channel proteins using electrochemical techniques. This study describes for the first time a method to produce high-impedance tBLM on a gold electrode using membrane fractions.…”

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

“…Individual insulin receptors reconstituted into a phosphatidylcholine (PC) membrane have been imaged, with height measurements confirming that the protein is oriented in two directions, with either the larger extracellular or shorter intracellular domains protruding out of the bilayer (Slade et al, 2002). This study illustrates the difficulty of controlling the orientation of the protein in supported membranes, although in some cases this challenge has been addressed by using specific labels (e.g., using histidine or streptavidin tethers) to immobilize the protein in one orientation (Elie-Caille et al, 2005;Giess et al, 2004;Trepout et al, 2007). A second practical limitation is that the conditions necessary for the formation of supported membranes from proteoliposomes may be significantly different than those for lipid vesicles, since the process of vesicle adsorption, rupture, and spreading is known to depend on the nature of the support as well as vesicle composition, lipid concentration, and temperature (Goksu et al, 2009;Johnston, 2007;Richter et al, 2006).…”