# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Peptide-tBLMs or Pep-BLMs
[207] have incorporated channel-forming peptides, _gramicidin_ and _alamethicin_, in a mercury-supported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled DOPC monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 3\({}_{10}\)-helical structure, which terminates at the N-terminus end by a sulfydryl group for anchoring to the metal [209], while the \(C\)-terminus end is covalently linked to the polar head of DMPE. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiopeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions, and to create a suitable environment for the incorporation of integral proteins. Nevertheless, in the conventional way of preparing tBLMs, both leaflets of the bilayer are formed independently through a \"bottom-up\" approach: first proximal layer by self-assembly process, and second distal layer completed (the most often) by fusion of vesicles. The building-up of pep-tBLMs has been mainly followed by SPR spectroscopy and FRAP [113, 210], and characterized by AFM, EIS, FM [210]. These studies report that a true lipid bilayer is not always fully obtained and that the films are not fluid on the micrometer length scale [210]. Actually, the step-by-step procedure of the \"bottom-up\" method sometimes leads to lateral heterogeneities or discontinuities in tethered lipid bilayers due to the partial surface coverage with some undisrupted liposomes adsorbed onto the solid surface [37, 71, 210]. Indeed, depending on their composition, vesicles could have a low propensity to fuse on the hydrophobic surface of the proximal layer exposed to aqueous solution, especially if they incorporate an integral protein [167]. In addition, the leaflet-by-leaflet formation process could lead, despite the successful reincorporation  
\begin{table}
\begin{tabular}{l l} \hline \hline
**Name** & **Amino Acid Sequence** \\ \hline P5 & Lip-Ala-Ala-Ala-Ala-CoAOH 1 \\ P7 & HS-(CH\({}_{2}\))\({}_{2}\)-Ala-Ser-Ala-Ala-Ser-Ala-CoOH \\ LP12 2 [FOOTNOTE:2]Footnote 2: Abbreviations: “Lip” for lipoic acid; “My” for myriotic acid (C14�������������������������������������������������������������������������������������������������������������������������������������������������������������������� \ \, }}}}\))\,\,\,\,\  
mentioned above, to a misfolding of the transmembrane proteins which require a lipid core to maintain their function; these latter can be denatured, even partially, during the coverage of the hydrophobic proximal layer by the distal monolayer and thus, lose their functional activity. Indeed, from a biochemical point of view, the only physical insertion of proteins, transmembrane or not, in a lipid bilayer is not a guarantee of the functional folding of the protein, and only a few report the functionality of large transmembrane proteins reinserted into tBLMs (It should be emphasized here that most of the reinsertion studies carried out with densely tBLM or stBLM are obtained from proteins which insert spontaneously into the preformed membrane from an aqueous solution) [115; 131; 185; 204; 206]. A possible explanation of this failure may be the small amount of reincorporated proteins [203], which does not allow to measure any protein activity nor quantify it. Lastly, the functionalization of the thiopeptides by the lipid anchor to generate thiolipopeptides requires a synthetic chemistry, often complicated, which limits the type of lipid anchor which can be added, classically dimyristoylphosphatidylylethanolamine (DMPE) [37]. It is the same situation with anchorlipids mainly constituted of two phytanoyll chains.