# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Polymer-tBLMs
This slowing-down effect of the immobilized tethers on the dynamics in the tethered bilayers has been confirmed by molecular dynamics simulation that revealed that the lateral diffusivities of both the proximal and distal layers are lower than for freestanding bilayers with PEG [78]. In addition, the lipid bilayer (membranes) formed on polymers often containsholes and defects [91]. A high grafting density and a long tether length induce undesired conformations, such as undulated tBLM systems, tether detachment, and even pores in the bilayer [78], which makes polymer-tBLMs generally less suitable for use as bio-detection platforms: the polymer acts as a passivation layer, which can greatly degrade the detection sensitivity on a noble metal such as gold [137], widely used in surface analysis methods (see Table 1), and the lipid membrane does not have sufficient electrical sealing properties for the investigation of ion transport process and quantitative characterization of ion channel activities [42; 51]. For these reasons and despite their potential attractiveness, only a few successful reincorporation of integral MPs has been achieved in polymer-tBLMs formed by \"bottom-up\" approach (i.e., using proteoliposomes for fusion on preformed proximal lipopolysaccharide layers during bilayer formation). They mainly relate to _cytochrome_\(b_{5}\)[20], _t-SNARE receptor_, a central player in membrane fusion in exocytosis and intracellular vesicle transport [138; 139], _lucteriorhodopsin_[136], _human platelet integrin receptor_ for modelling of cell adhesion processes [140], and the multi-drug resistance protein 1 (MDR1) for which drug interaction in vitro assays (pravastatin) have been carried out [141]. It is likely that the two-step process of forming, one after the other, the two membrane leaflets is unfavorable for the preservation of functional MP structure during bilayer formation and hence, a new \"top-down\" approach have been developed (see below Section 4.5). On the other side, polymer-tBLMs are able to respect the deformability or the natural fluctuation of the membrane and they can provide a more natural and cell-like surrounding than a solid surface [97]. By modifying the lipopolymer into \"lipoglycopolymer\", it becomes possible to reproduce by mimicking, the cytosol or cytoskeleton of the cell [142]. For these reasons, polymer-cushioned and polymer-tBLMs have been widely used, as models of the extracellular matrix or the cell surface glycocalyx [127]. More recently, they have been further developed in _polymer-tethered lipid multi-bilayers_ formed by a stack of three or four bilayers separated from each other by covalently attached PEG molecules in surface of each membrane. This multi-bilayer stacking results in a lesser influence of the tethered-lipopolymer than in polymer-tBLMs. Bilayers within stacks are free of optically visible defects and show a good stability. Lateral diffusion can be adjusted by the number of bilayer in the stack, with a correlated gradual increase of lipid diffusion with the degree of bilayer stacking [60]. This tunable diffusion and mechanical properties made such multi-bilayers systems cell surface-mimicking materials with well-defined surface properties for the analysis of cellular mechanosensitivity with advanced breakthroughs in the understanding of cell adhesion and cell migration, as recently shown for polymer-tethered lipid multi-bilayers functionalized with N-cadherin linkers [143] (for details see [60]). However, such applications of polymer-tBLMs is out of the scope of this review focused on the membrane reincorporation of functional integral membrane proteins and therefore they will not be discussed further.