# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Sparsely-tBLMs or stBLMs
While _alamethicin_ is not able to penetrate _fully tethered lipid bilayers_, in sparsely tethered ones, individual peptides may first adsorb to the membrane surface and then aggregate before inserting into the bilayer to form pores [110]. An interesting review introduced the different applications of numerous cholesterol-based anchors and tethers, even beyond the field of membrane protein reconstitution (see [158]). More recently, [PERSON] et al. have synthesized a series of new bifunctional cholesterol compounds with cyclic sulfide as surface linker chemical group [89]. Interestingly, they proposed cholesterol derivatives with an electroactive group to study electron transfer processes, or fluorescently-labeled to monitor cholesterol trafficking in membranes. Another tethering chemistry has also been explored to form sparsely-tBLMs. The tethering monolayer (i.e., proximal leaflet) is prepared with two benzyl disulphide families, one being a tethering group comprising an eleven oxygen-ethylene glycol linker group with a single C20 hydrophobic phytanyl chain as the hydrophobic tether, named as DLP (\"double-length\" reservoir phytanyl lipid (DLP) [188]), and a second being a spacer molecule containing a four oxygen-ethylene glycol spacer, terminated with an OH group as diluting molecules [116], generally used in a 1:9 ratio, respectively. The bilayer is then completed by rapid solvent exchange of an organic solution of phospholipids, which allows to tailor the lipid composition as desired to mimic natural cell membranes [99; 100; 189]. These types of stBLMs have revealed good sealing properties [24] and provide a large ionic reservoir and space for transmembrane protein insertion [188]. They appear particularly suited for EIS investigations. Hence, they have been intensively used (i) to study ion channel activity or conductance, like _alamethicin_ a small peptide voltage-gated ion channel [190], a synthetic ligand-gated ion channel (SLIC) [24], small peptide ionophores, like _alinomycin_ or _gramicidin_[117], the _chloride intracellular ion channel_ (CLIC1) a metamorphic protein able to insert into phospholipid bilayers enriched in membrane sterols from their soluble state [191], or (ii) to identify the mechanism of the antimicrobial peptides forming different types of pores in lipid membranes [192], like _Kalata B1_ and _Kalata B2_, cyclic antimicrobial and insecticidal cyclotides [193], or _Melimine_[194]. They have been also used as harboring lipid supports for cell-free expression (see Section 5) and functional reconstitution of recombinant _OprF_ porin as target for antibacterial drugs, and the subsequent structural study by NR [195]. More recently, they have been employed to investigate the effect of the heat transfer between cell membranes and gold nanoparticles (GNPs) used in laser-targeted treatment for infections, tumors and for the controlled released of drug in situ [196]. They also represent a platform as substrate for phospholipase A (PLA) catalytic assay [197] or for toxicity examination of food cyanobacteria toxins like _micricystins_, suspected to compromise the cell membrane integrity [198]. Finally, with the goal of avoiding the cumbersome chemistry of anchor thiolipids synthesis, [PERSON] et al. have recently developed a versatile approach based on wet chemistry via aryldiazonium sulfonic acid, for grafting PEG as cushion mixed with a commercial surfactant, i.e., Brij58 as alkyl chain harpoon, for subsequent formation of tBLMs by adsorption and spontaneous fusion of lipid vesicles [103]. In this stBLM, the lipid bilayer was stabilized by molecular coatings composed of diluted anchor-harpoon surfactants that grab the membrane with an alkyl chain out of a PEGylated-hydrogel layer, which acts as a soft hydration cushion [199].