# 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
#### 4.5.2 Using Biotin/Avidin as Spacers  
Another strategy for forming tBLMs by liposome spreading uses the affine avidin/biotin system as spacer for tethering the membrane (Figure 3b). In a first step, intact biotinylated vesicles are accumulated by affinity on a sublayer of immobilized streptavidin. As liposomes do not fuse spontaneously on soft matter, the formation of the bilayer is triggered by addition of soluble poly(ethylene glycol) (PEG), a fusogenic agent of lipid vesicles [118; 214] or by replacing the medium by a hypotonic solution [228]. One of the crucial steps in the formation of tBLMs is the achievement of vesicle fusion on the template, in such a way that a continuous fluid bilayer without defects can be produced in a reasonable time, compatible with the preservation of the native membrane proteins. The formation of a tethered bilayer on a streptavidin sublayer in this \"top-down\" assembly makes it possible to overcome the constraints imposed by uncontrolled kinetics of spontaneous vesicle fusion since this latter requires triggering [81; 217]. Additionally, since no particular anchorlipid is needed in this strategy, this allows the lipid composition to be modified as desired. Thus, by using this system, [PERSON] et al. [134] have successfully tethered and fused phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane and containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The overall process and the triggered fusion of the immobilized (proteo-)liposomes have been monitored by SPR and FRAP, respectively. A homogenous flat surface of soft material corresponding to a biomimetic (proteo-)lipid bilayer anchored to the solid substrate was obtained [81; 134]. [PERSON] et al. [228] used the avidin-biotin interaction to tether lipid bilayer containing photosynthetic antenna proteins (light-harvesting complex 2, LH2- and light-harvesting core complex, LH1-RC) for functional analysis. The energy transfer--obtained from LH2 to LH1-RC within the tethered membrane and analyzed by fluorescence spectroscopy--has been ascribed to the diffusivity of the proteins inside the bilayer. Such a result indicates that biotinylated planar membranes tethered by pillars of avidin molecules and formed by spreading of proteoliposomes in a \"top-down\" approach, offer the possibility of reconstituting in vitro large photosynthetic complexes and mimic a natural situation. As demonstrated through these examples, the main advantage of forming tBLMs by vesicle fusion incorporating only 0.5-1 mol% of biotinylated-phospholipids (e.g., DMPE, DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, or DOPC) lies in the possibility to complexify the membrane lipid composition [217] or even to be able to use extracted membrane lipids [134]. Finally, the affine avidin/biotin system is commonly used in the design of microarrays. Avidin/biotin-tBLM arrays have been built in a parallel microfluidic system (i.e., in parallel microchannels) using the \"top-down\" approach, and used for multiplexed label-free analysis of lipid-protein interactions by SPRi. Multiplexed analysis of protein interactions has been demonstrated with several systems, including monosialoganglioside (GM1) receptor for cholera toxin (CT) and phosphatidylinositol phosphate (PIP) lipid for PIP-binding proteins, for which a calibration curve was obtained. The tethered membrane array technology, in combination with _surface plasmon resonance imaging_ (SPRi), offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with MPs embedded in the near-native environment [217].