# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Sparsely-tBLMs or stBLMs  
In order to address issues of space and hydration of the sub-membrane compartment to better facilitate protein incorporation into tBLMs, _sparsely tethered-bilayer lipid membranes_ or stBLMs have been developed [125]. The density of the tethered anchor thiolipid can be reduced, either by using a longer and bulkier disulfide (or lipoic acid) surface anchoring segment instead of thiol [77; 101], or by diluting the anchor thiolipid forming the proximal leaflet with a second shorter surface-active backfilling molecule such as tetraethylene glycol lipoic acid ester [159], thiol terminated tetraethylene glycol molecules [85] or \(\beta\)-mercaptoethanolhanol (\(\beta\)ME), which competes for space by co-adsorption on the substrate [94; 102]. For sparsely tethered membranes diluted with \(\beta\)ME, a hydration level of up to 75 vol% of the submembrane compartment has been achieved, depending on the tethering density of the distal leaflet and the type of lipid used to complete with the outer leaflet [94]. However, the sealing properties of the resulting lipid membrane are significantly reduced with the increase of molar % of backfillers, likely due to the high level of hydration leading to a higher propensity for defect formation [94]. Additionally, the exact composition of its inner leaflet, depending on the relative solubility of the compounds (i.e., anchor lipids and backfiller molecules) and their kinetic binding, is no longer known [125], which is not the case when using a thiolipid with a modified anchoring group [77; 101]. However, highly hydrated systems that are stBLMs formed by dilution strategy appear optimal for the study of membrane properties that are not related to its electrical sealing properties, because a very fluid (or disordered) bilayer is created with high hydration levels [88], suggesting the presence of significant submembrane space for protein incorporation. Indeed, it has been demonstrated that stBLMs are a better option for protein reconstitution [124] than _densely tethered-BLMs_, presumably because of a higher flexibility of the membrane due to the possible presence of free phospholipids in the proximal layer [88; 151]. To assemble stBLM, new class of thiolipid anchors have been prepared by changing the two branched chains (phytanoyl) by saturated (C14 \(n\)-alkyl [94] or C16 \(n\)-alkyl [154] chains) or unsaturated acyl chains (C18:1 [96]. Coadsorbed with a short-chain backfiller (i.e., \(\beta\)ME), this variety of chemical structures forming the distal leaflet makes it possible to refine the properties of the resulting membrane architecture with the possibility of supplementing the distal leaflet with various phospholipids (e.g., DPhyPC: 1,2-diphytanoyl-\(sn\)-glycero-3-phosphocholine, DOPC: 1,2-dioleoyl-\(sn\)-glycero-3-phosphocholine, POPC: 1-palmitoyl-2-oleoyl-\(sn\)-glycero-3-phosphocholine, and DMPC: 1,2-dimyristoyl-\(sn\)-glycero-3-phosphocholine [94], including charged lipids such as DOPG: 1,2-dioleoyl-\(sn\)-glycero-3-phosphoglycerol [154] or mixed Cholesterol/DOPC) [153; 177]. In addition to supplying a more spacious sub-membrane compartment containing water, stBLMs completed with a distal leaflet composed of unsaturated phospholipids (DOPC) have been shown to be more fluid than _fully tethered membranes_, with diffusion coefficients in the distal leaflet, approaching values similar to the natural cell membranes (diffusion in the proximal leaflet is hindered by the presence of the anchorlipids) [88]. Likewise, stBLMs formed with an anchor thiolipid containing double bonds in the alkyl segment exhibit higher lipid diffusivity relative to those formed with saturated alkyl chains. This increased membrane fluidity is due to the more disordered bilayer structure caused by the more flexible unsaturated alkyl chains [96]. This further demonstrates the high flexibility with which membrane properties can be fine-tuned depending on the desired application.