# 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
(**a**) using PEG as spacers, by fusion of liposomes containing either DSPE-PEG-NHS or DSPE-PEG-PDP on an amine-coated surface; (**b**) using biotin/avidine as spacers, by fusion of biotinylated vesicles on a sublayer of immobilized streptavidin. In both cases, the liposome fusion can be triggered by a fusogenic agent (i.e., PEG-8000 solution [81; 134; 214; 217] or an amphipathic \(\alpha\)-helix (AH) peptide [39]). DSPE-PEG-NHS: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide; DSPE-PEG-PDP: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate]. steric hindrance for reincorporation of membrane proteins having large extra-membrane domains [51]. Another important point when studying integral protein concerns the lipid composition of the bilayer. Indeed, it is now well-known that the lipid environment has an impact on the proper functioning of membrane proteins [8; 9; 10; 218; 219]. Ideally, MPs should be investigated in their native environment. Thus, the lipid composition of tBLMs must be as close as possible of the one of the natural lipid membrane in which the membrane protein is embedded. A particular attention should also be paid to the importance of non-bilayer lipids (Non-bilayer lipids can be defined as cone-shaped lipids with a preference for non-bilayer structures with a negative curvature, such as the hexagonal (HII) phase) [220; 221] in the functional activity of intrinsic MPs. These lipids are present in large amounts in all membranes, and especially in energy-converting membranes where they constitute about half of their total lipid content [222]. Due to their conical shape, they alter the lateral pressure profile in the lipid bilayer--called _lateral pressure model_ (LPM)--and generate elastic energy of curvature--called _flexible surface model_ (FSM). Hence, they contribute to the structural flexibility and plasticity of membranes by stabilizing regions of high curvature or packing around the hydrophobic core of MPs [223]. Their role in membrane functioning and their impact as regulators of functional MP activities are now widely described [15; 16]; they should be much more taken into account in the lipid composition of membrane models than they are currently. Until now, the compositional simplicity of biomimetic membrane models in general, often limits the level of knowledge that can be gained about the structure and the function of transmembrane proteins. Recently, [PERSON] et al. [224] have explored a generic methodology wherein \"_native cell-membrane vesicles_\" (NMVs) are merged with a synthetic vesicle containing POPC and PEGylated lipids that promote vesicle fusion. These hybrid vesicles readily spontaneously rupture into a continuous polymer-supported membrane, which contains essentially all the natural components of the plasma membrane of the cell line from which the NMVs are generated. By using the \(\beta\)-_secretase 1_ (BACE1), a transmembrane protease--overexpressed in an insect cell line and exhibiting asymmetricoplasmic and cytoplasmic extra-membrane protruding regions which can be probed independently--the authors demonstrated that the mobility and activity of transmembrane proteins are preserved. This \"top-down\" approach corresponds to the first report demonstrating the lateral mobility of a transmembrane protein in a _polymer_-tBLM derived from the cell membrane, with a large (>1 nm) protruding region facing the substrate. It provides new insight into how biomimetic surfaces can be fabricated from native membranes, for fundamental studies of membrane biophysics and production of surface-based analytical sensor platforms [224]. For the sake of completeness, we can cite the works of [PERSON] et al. [93], who propose an intermediate solution between the \"bottom-up\" and \"top-down\" approaches, with a pre-functionalization of the surface with a lipopolymer tether and a subsequent fusion of (proteo-)liposomes to form continuous tethered polymer-supported membranes.