# 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  
Another strategy to improve space and hydration in the sub-membrane compartment is based on the self-assembly of thio-polar peptides as spacers to form _peptide-tethered-bilayer lipid membranes_ (pep-tBLMs). These pep-tBLMs have been shown to be suitable for membrane protein reincorporation by providing a biocompatible sub-membrane environment in which extra-membranous subunits can fold into a native-like conformation [71]. Peptides are actually of the same nature as the cytoskeleton inside or the extracellular matrix outside the cell with which the protuberance of the membrane proteins can interact in real life. The main advantage of using peptides as tethers is that their length, their secondary structure and their hydrophilic properties can be easily tuned by changing their aminoacid sequences. This enables a flexible adjustment of both membrane-substrate separation and viscosity of the tether units, which are important to ensure sufficient lateral membrane diffusivity for functional protein incorporation [200]. Thus, pep-tBLMs are well-designed to preserve the integrity and functionality of membrane proteins. On the contrary, the resistance of pure pep-tBLM is rather low, so these membranes are not particularly suitable for electrochemical and EIS measurements [71]. Classically, peptides used as tethers to form pep-tBLMs are prepared from synthetic or native diopeptides, or thiolopeptides (Table 2, [71]), used without backfilling molecules (no dilution). These peptides are functionalized at their N-terminus by a sulfur group such as a cysteine, or a lipoic acid designed for self-assembly on gold, and except for LP12 which can be used without additional chemistry, their _C_-terminus extremity is chemically activated afterwards for the coupling of the amino polar headgroup of different phosphatidylethanolamine (PE), thus forming the proximal lipid monolayer [37], which acts as the anchor of the membrane by forming the proximal leaflet of the bilayer [111; 115; 131; 201; 202]. As for anchorlipids, pep-tBLMs have been firstly formed by \"bottom-up\" approach [34]. The first peptide layer was built via self-assembly process from a dilute solution of tether molecules. The bond with the substrate provided stable anchoring of the proximal leaflet. After chemical activation and grafting of PE via an ester bond, the distal layer was generally formed by fusion of liposomes with or without the reconstituted protein of interest onto the hydrophobic self-assembled proximal layer [72]. Different integral membrane proteins e.g., cytochrome c oxidase [131], cytochrome bo3 ubiququinol oxidase [203], H+-ATPases (from chloroplasts and E. coli) [115, 201, 202, 204], dimer of nicotinic acetylcholine receptor from Torpedo californica [111], hERG potassium channel [205], integrins [206] or channel-forming peptides like gramicidin or alamethicin [207] have been successfully reincorporated in pep-tBLMs formed with functionalized thio(lipo)peptides. Pep-tBLMs have also been developed by using combined Langmuir-Blodgett and Langmuir-Schaefer transfers [208]. In this approach, a monolayer of 1,2-dimyristoyl-sn-glycero-3-phospho-_ethanolamine_ (DMPE) is directly transferred by vertical dipping onto activated carboxyl groups of a P19 self-assembled monolayer formed on gold by strong S-Au interaction. The distal lipid monolayer is obtained by Langmuir-Schaefer transfer. Owing to this stepwise assembly, it is easy to change the lipid composition of the distal layer. By this way, the adsorption of the amyloid \(\beta\)-peptide (A\(\beta\)40) with several lipid mixtures has been analyzed. The A\(\beta\) adsorption was found critically depending on the lipid composition of the membranes, with A\(\beta\) specifically binding to membranes containing sphingomyelin. Further, this preferential adsorption was markedly amplified by the addition of sterols. [PERSON] et al.