# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Anchorlipid-tBLMs
For these reasons, they have been extensively used to study small membrane-active peptides, such as antimicrobial peptides [149; 150], pore-forming toxins such as gramicidin [41; 164; 123; 104] or large oligomeric \(\alpha\)-hemolysin [165; 166], ion channels [167], such as valinomycin [168; 169; 164; 160; 161; 164; 166; 167] or ligand-gated ion channels [170]. They have been also used as a bilayer platform to study the structure of the _human immunodeficiency virus type_ 1 (HIV-1) _Gag protein_ as well as its interaction with lipid bilayers [171], to mimic the outer membrane of Gram-negative bacteria which can cause some of the most dangerous multi-drug resistant infections [150; 151; 172], to investigate membrane incorporation from aqueous solution of bulky proteins such as _OmpF_ porin from _E. coli_[173] and micelles of Triton X-100 solubilized _human ether-a-go-go-related gene_ (hERG) _potassium channel_[174], or for biosensing applications [166; 41; 42]. Nonetheless, in most cases, the chemical structure of the tethers has a significant impact on the structure of the resulting lipid bilayer [166] and the functional incorporation of membrane components. For the important class of anchorlipids, i.e., _telechelics_, neutron scattering [32; 77] and PM-IRRAS studies [105] have revealed a poorly hydrated spacer segment despite polyethylene glycol (PEG) being water soluble, due to a coiled (helical) conformation exposing the hydrophobic carbon segments of the ethylene glycol moieties to the surroundings when assembled into tBLMs, excluding water from the sub-membrane space [105]. Sub-membrane hydration can be changed by modifying the spacer segment and increasing the number of ethylene glycol units. However, increasing the length of the tethers also leads to a significantly higher number of defects [77]. In addition, the tether density affects the resulting bilayer properties [102]. The high packing density of the tethers results in a densely tethered proximal leaflet, which, in addition to limiting hydration in the surrounding medium, imposes spatial constraints which may have an impact on the structural adaptation of the sub-membrane protein subunit. Since the incorporation of proteins with bulky extra-membrane domains requires space and significant hydration environment, thiolipid anchored-tBLMs from _telechelics_, due to their high packing density, have been shown to be detrimental to harbor large trans-membrane proteins [71; 175]. As recently confirmed by molecular dynamics calculations, the tether density of thiolipid anchors affects the hydration level, bilayer fluidity, defect density, free energy of lipid flip-flop, lateral mobility and membrane dielectric permittivity [176]. Therefore, it has been found that pure thiolipid anchored-tBLMs, also named _densely_ or _fully tethered-BLMs_, partially respond to the challenge of loss of protein diffusion or induction of protein denaturation due to friction with the fully tethered proximal lipid layer, which can hamper functional incorporation of bulky trans-membrane proteins.