# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 2 Design of Tethered-Bilayer Lipid Membranes  
tBLMs are a natural progression from the planar supported lipid bilayers (SLBs), first reported by [PERSON] et al. [19]. SLBs, classically obtained by the spreading of small unilamellar vesicles on hydrophilic solid supports [45; 46; 47], including glass, mica, titanium and silicon oxides, or gold (for recent reviews see articles by Lind & Cardenas 2016 [48] and [PERSON] et al. [40]), consist in a lipid bilayer deposited and separated from the solid substrate by an ultrathin film of water (0.5-2 nm) [45; 49; 50; 51]. This aqueous layer acts as a lubricant and confers to SLBs the fluidity required for lateral diffusion in 2D space [45]. In this model, lateral and rotational mobility of individual lipids are preserved and anything linked to the phospholipids or glycolipids in the upper leaflet retains this mobility [52]. Given this key feature, SLBs have been used extensively over the past decades to study the spatially and temporally regulated lipid-lipid or lipid-protein lateral interactions [53; 54; 55; 56], lipid segregation [57; 58; 59], protein clustering and cell adhesion [60; 61; 62; 63] and membrane dynamics [64; 65; 66]. However, the close proximity of SLBs to the substrate affects the diffusion of lipids and proteins, which is more than twice slower than in free-standing bilayers under the same conditions [67]. This limitation is due to the fact that the substrate exerts a greater influence on the behavior of the proximal leaflet than the distal leaflet of the SLBs, due to its closer proximity to the surface [68; 69; 40]. The roughness of the substrate and the complementarity between the surface and lipid charge will determine the magnitude of this surface influence [50]. Furthermore, SLBs suffer from the crucial drawback of not possessing a large hydration reservoir on both sides of the membranes, which limit examination of transmembrane proteins. The fundamental requirement for a membrane protein to function properly is to be surrounded by buffered-saline solution on both sides of membranes [70]. In SLBs, hydration layer is often not thick enough for proper folding of large extra-membranous domains of transmembrane proteins, which can extend to several tens of nanometers far out from the bilayer [18]. The limited membrane-substrate distance, which can lead to strong non-physiological interactions with the solid support [18; 20], can cause both a loss of protein dynamics and a partial loss of its functionality, or even complete denaturation of the protein [71; 72; 73]. In addition, anionic substrates (such as quartz, mica, silica, silicon oxide) may hinder (in the absence of divalent cations) the formation of SLBs enriched in negatively charged lipids in the proximal leaflet, due to electrostatic repulsions [46; 66; 74]. However, negatively charged lipids, like phosphatidylcholine (PG), phosphatidylcholine (PI), cardiolipin (CL) or even lipopolysaccharide (LPS) in the Gram-negative bacteria membrane, are important signaling lipids which can trigger membrane protein association and affect membrane-regulated pathways. They are essential in membrane function, and studies of the membrane phenomenon regulated by these lipids are becoming crucial for a realistic understanding of membrane-related events. The use of mimetic sample systems with ever greater biological precision in lipid composition is now required. For all these reasons, more advanced planar model membranes are currently in development with the aim to create more accurate biomimetic systems adapted for integral (trans-)membrane protein characterization, where the substrate interactions are minimized and large solution reservoirs on both sides of the bilayer are provided. In tBLMs, the lipid bilayer is separated from the surface of the substrate by insertion of a soft and flexible hydrophilic layer of \"tethering\" molecules that anchor the proximal leaflet [31; 34; 71; 72; 75; 76].