# 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
By developing an original test involving the peculiar adsorption property of a cytoplasmic protein, the _nucleoside diphosphate kinase-B_ (or NDPK-B) to associate in a discriminatory manner to nude gold surface or anionic lipid membranes, it was shown that the pep-tBLMs is formed without defects [200]. The main original feature of this methodology, compared with the classical way to form pep-tBLMs, is that it is based on the use of preformed entire vesicles before fusion, and as we saw before, the lipid composition of the bilayer can be thus easily tuned and made more complex (Table 3), or even formed from natural membrane lipid extracts (unpublished data). For instance, some non-bilayer lipids, like PE, can be added in the lipid composition (Table 3). Due to its conical shape, this fusogenic lipid preferentially forms non-bilayer phases and promotes inverted hexagonal (HII) phase intermediates that can favor membrane fusion [223; 238; 239]. Hence, the lipid composition can be readily adapted for (trans)membrane protein reinsertion since it offers the possibility to reincorporate them in a native-like lipid environment, which is essential to keep their natural conformation and their biological activity on a gold surface for performing ligand binding assays. Therefore, the fusion of a proteoliposome of complex lipid composition, produced by cell-free expression and inserting a functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs) of high pharmacological interest, led to an efficient reinsertion of this bulky membrane protein exhibiting extra-membrane subunits in the planar pep-tBLMs (Figure 4), while retaining its lateral mobility [200]. (_GPCRs are an important class of membrane receptors implied in numerous diseases and cellular dysfunction with a great relevance in pharmaceutical research and drug screening applications. CXCR4 represents an important therapeutic target involved in cancer cell migration and in HIV-1 entry_). The functionality of the reincorporated receptor was assessed by ligand binding assays using SPRi, and revealed that the receptor retains both its native folding and its proper orientation in the planar pep-tBLMs, suggesting that the opening of the proteoliposomes induced by the AH peptide followed a \"parachute\" mechanism. This successful result could probably also be ascribed to the fact that proteoliposomes were produced by cell-free expression that allows a vectorial insertion of proteins in the membrane directly in a functional orientation (seeSection 5). The possibility to create pep-tBLMs inserting the functional GPCR in a microarray format by spotting P19-4H on micropatterned support for multiplexed drug screening has been also demonstrated [43]. Due to being based on peptide tethers, this versatile approach can be considered for the reconstitution of other membrane proteins and even, to study the influence of the peptide composition of the sub-membrane compartment on the functionality of the reconstituted integral MPs, as one can see it in natural cell membranes. Figure 4: (a) pep-tBLM obtained by “top-down” approach, integrating a functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs) of high pharmacology interest. The functional proteoliposome has been previously obtained by a cell-free expression system in presence of pure liposomes; (b) side (A) and top (B) views of the binding site structure of functional CXCR4 inserted in the pep-tBLM with domains and aminoacid residues (highlighted in purple) involved in the specific binding of T22, an antagonist ligand which specifically inhibits human immunodeficiency virus type 1 (HIV-1) infection mediated by CXCR4. This structure reveals that the binding site of T22 is localized outside the membrane and that 5 domains need to be correctly folded for a functional activity.