# 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
#### 4.5.3 With Peptide as Tethers  
An alternative approach to take advantage of pep-tBLMs benefits which truly mimics the intra- (i.e., cytoskeleton) or the extra- (i.e., extracellular matrix) protein nature of the membrane environment, and to form the bilayer in a single step, but avoiding any chemistry, has been proposed. In this approach, liposomes including or not reconstituted membrane proteins are attached by a metal-chelate interaction to peptide spacers grafted on gold surface, before triggering their fusion by a fusogenic agent to form pep-tBLMs (Figure 2a) [200; 229]. The metal-chelate interaction has been already used to graft MPs onto chemically modified gold surface via the affinity of their histidine-tag for a nickel-chelating nitrilotriacetic acid (NTA) surface before reconstituting them into the lipid environment (see below Section 4.6) [107], but never for attaching the lipid membrane onto grafted peptide spacers. For this purpose, a new peptide spacer derived from the natural thiopeptide (P19) and modified in its \(C\)-terminal extremity by four histidine residues (P19-4H, Table 2 and Figure 2b) to bind liposomes containing 2% of a chelating lipid (DOGS-NTA-Ni: 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxymethyl)iminodiacetic acid)succinylyl] (nickel salt)) (Figure 2c) has been recently designed [229]. Among all the peptides spacers already used to form pep-tBLMs, P19 was chosen since its N-terminal cysteine residue allows spontaneous grafting on gold surface via stable Au-thiolate bond formation, making it one of the most widely used for that purpose. As it is a water-soluble peptide derived from the subunit \(\alpha\) of laminin, an extracellular matrix glycoprotein involved in the cell-cell interaction and cell differentiation, its natural composition offers an adequate hydrophilic sub-membrane environment for membrane protein reinsertion [37; 203; 205; 206; 230]. DOGS-NTA presents a chelating headgroup that forms a coordination complex with histidine residues in the presence of nickel with a high binding affinity (\(K_{d}=10^{-13}\) M for a six-residue polyhistidine tag at pH 8.0) [231]. Suitable to coat materials, chelating lipids have been used to immobilize proteins and functionalize different systems, like lipid surfaces for bioreceptor immobilization [232; 233]. After tethering, liposome fusion is triggered by a fusogenic agent to obtain a planar membrane, as previously proposed for other systems (see above). In the present approach, an amphipathic \(\alpha\)-helix (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein [234] was used to induce vesicle rupture, as already demonstrated for formation of tBLMs on polymer (PEG) cushion [39] or on mesoporous silica [213]. The main reason of this choice is because after promoting vesicle swelling and rupture, this peptide desorbs from planar bilayer, leading to the formation of a pure lipid bilayer exempt of any \"extra\" molecules [235; 236; 237]. The mode of attachment by chelation between P19-4H and the headgroup of DOGS-NTA proposed in this \"top-down\" method has conferred a great stability during handling compared to other pep-tBLMs described in the literature, in which the lipid bilayer is only anchored by hydrophobic interaction (i.e., hydrophobic chains of DMPE attached to the \(C\)-terminal end of peptide spacer inserted inside the bilayer, see Section 4.4), possibly leading to a delamination and/or partial destruction of the membrane architecture with formation of defects in the bilayer structure. FRAP and AFM studies have confirmed that using this \"top-down\" approach, the bilayer obtained with or without membrane proteins is homogenous, fluid, continuous and the lateral mobility of membrane compound constituents are preserved.