# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Polymer-tBLMs  
To overcome the problem of the close proximity between membrane and support, polymer-cushioned membranes have been developed, pioneered by [PERSON]'s group [21]. This model allows separating membrane from the solid substrate using a soft polymeric material that rests on the substrate and supports the membrane [127]. The hydrated polymer used to \"cushion\" or \"tether\" the supported membrane acts as a lubricating layer between the membrane and the substrate. This approach significantly reduces the frictional coupling between the transmembrane-incorporated proteins and the solid support, and hence the risk of protein denaturation. Depending on the type of fabrication method and polymer system used, the addition of the polymer layer can result in bilayer-substrate distances of 5-100 nm, making these model membranes suitable for the analysis of transmembrane proteins. Several different strategies for assembling lipid bilayers supported by a polymer have been previously pursued [127; 128]. One relatively straightforward fabrication approach relies on the application of polyelectrolytes as cushion materials and the subsequent addition of a bilayer containing a certain fraction of lipids with oppositely charged headgroups. The attachment of the bilayer on the surface of polyelectrolytes using attractive electrostatic interactions can be combined with the established method of layer-by-layer (LbL) deposition of polyelectrolytes, which provides flexibility in terms of accessible polymer cushion thicknesses. However, the presence of polyelectrolytes in electrostatically stabilized polymer-supported lipid bilayers may have a disturbing effect on the properties of reconstituted membrane proteins [60]. An alternative method has emerged consisting of the stable attachment of the lipid bilayer to the polymer cushion by tethering. In the resulting supramolecular assembly, referred to as _polymer-tethered lipid bilayers_, lipids with macromolecular head groups, so-called _lipopolymer tethers_, are incorporated into the lipid bilayer [33; 34]. In this system, the proximal lipopolymer layer is generally first attached to support by chemisorption and the bilayer is completed with a distal lipid leaflet, formed either by vesicle fusion or Langmuir-Schaefer transfer, in a two-step preparation process also called the \"bottom-up\" method (for details, see [60; 128]). The polymer head groups act as spacers that control the substrate-membrane distance and, in common with polymer cushions, prevent direct contact between extra-membraous transmembrane protein subunits and the bare substrate surface [60]. The ability to flexibly adjust both the length of the polymer spacer and its lateral density allows fine adjustment of the membrane-substrate distance and the viscosity of the polymer layer, both of which control the lateral diffusivity and function of transmembrane proteins. Thus, the separation distance in polymer-tBLMs is enough to host transmembrane proteins [20]. Additionally, it was shown using _polarization modulation-infrared reflection absorption spectroscopy_ (PM-IRRAS), a surface sensitive technique applicable to thin film [135], that polyethylene glycol (PEG), conventionally used as the tethered-polymer, adopts an amorphous, disordered conformation, which promotes hydration of the inner layer essential for the proper functioning of reinserted transmembrane proteins. It forms a water-rich region between the bilayer and the metallic surface with a water content up to 90 \(\pm\) 3% as determined by NR [97]. However, the nature and the density of the attached lipopolymer may significantly impact the membrane properties. In particular, the lateral diffusion of both free lipids and embedded membrane proteins can be hindered by a tethering density greater than 4-5% to 10% depending on the nature of the lipopolymer [20; 136; 91], with the possible presence of motionless fractions [90].