# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Vesicle Fusion Method to Form tBLMs: A \"Top-Down\" Approach
#### 4.5.1 Using PEG as Anchoring Spacer  
In this polymer-coated liposome spreading procedure, the bilayer is achieved by deposition of vesicles containing a mixture of PC and either DSPE-PEG-NHS (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethylenediyl)-N-hydroxysuccinimide) [35] or DSPE-PEG-PDP (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylenediyl)-2000-N-[3-(2-pyridyllithio)propionate]) [39], which act as both spacer and anchor molecules. These spacer lipids are terminated by an activated group, which reacts with any amino-coated surface (cysteineamine-coated gold or amino-silanized glass) or gold surface, respectively (Figure 3a). Once the vesicles are bound to the surface, their rupture, possibly triggered by a fusogenic agent [213], leads to the formation of the bilayer. Formation of these _polymer-tBLMs_ has been followed using SPR spectroscopy [35; 36; 130], QCM-D [39] and characterized by AFM [36; 83]. They can be used to reconstitute raft lipid domains in the tBLMs in a microarray format [83]. Studies of the diffusion coefficient of individual lipids within the bilayer by FRAP measurements have indicated that the bilayer is fluid and continuous to preserve the high lateral mobility of membrane constituents despite the proximity of the solid support [35; 36; 130]. This type of bilayer has been successfully used to investigate the protein/membrane interactions of different kinds of integral proteins such as the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane [36] or the _aquaporin_\(Z\) (AqpZ) [39]. By using a sophisticated model mimicking cellular compartmentalization obtained thanks to polymer-coated liposomes spreading associated with distinct protein markers of intra- and extra-cellular milieu [130], [PERSON] and her group have succeeded in studying for the first time, the translocation of the adenylate cyclase, a bacterial toxin produced by _Bordetella pertussis_ (CyaA) through a lipid bilayer [129]. It should be noted that this last contribution is a remarkable example of the usefulness of tBLM models to understand molecular mechanisms that occur at the level of plasma membranes, since it allows a real breakthrough in the understanding of the protein translocation and transport across biological membranes under precisely defined conditions [215; 216]. Figure 2: (**a**) Principle of formation of pep-tBLM by “top-down” approach onto peptide tethers. The pep-tBLM is obtained after vesicle fusion of liposomes containing 2% of a chelating lipid DOGS-NTA-Ni, tethered onto P19-4H peptides, grafted themselves on gold surface via stable Au-thiolate bond; the vesicle fusion is triggered by an amphipathic \(\alpha\)-helix (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein; (**b**) 3D modelling of P19-4H; the three characteristic parts of the peptide are represented with the cysteine residue in red for the grafting on the gold surface, the central \(\alpha\)-helix in green for the spacing and the 4 Histidine residues in blue for the attachment of liposomes containing DOGS-NTA by chelation in the presence of nickel salt. The sequence of P19-4H and P19 peptide from which P19-4H has been derived are indicated below; (**c**) Structure of the DOGS-NTA-Ni able to interact specifically with P19-4H (adapted with permission from [200]. Copyright 2017, American Chemical Society). An important advantage of the direct incorporation of the spacers in vesicles is the possibility to control their percentage. This provides a spacer layer with a density lower than the fully tethered proximal leaflet formed by self-assembly of the _telchelics_[30; 104] or even lipopolymer [91]. As seen above, a high tether density leads to a poorly hydrated sub-membrane compartment which adversely affects the membrane fluidity and constitutes a  
Figure 3: tBLMs formed by fusion of lipid vesicles containing both the anchor and the spacer molecules on a functionalized surface via a “top-down” approach.