# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Protein-tBLMs or ptBLMs  
In most tBLMs previously described, and allowing the incorporation of MPs, the protein orientation in the membrane is rather casual; the addition of either solubilized channel proteins or the fusion of proteo-liposomes results in the spontaneous incorporation of proteins into the tethered membranes, but does not allow for the desired control of their orientation [72]. Indeed, if one of the two extramembraneous domains of the protein is much bulkier than the other, incorporation in a tBLM occurs preferentially with the bulkier domain turned towards the aqueous phase, in view of the limited space of the hydrophilic sub-membrane compartment of the tBLMs. Thus, it is extremely difficult to control the insertion and this random orientation may prevent the functional analysis of the protein. Moreover, the surface concentration of the reconstituted protein in the lipid bilayer is not well controlled. To overcome these problems, another strategy has been proposed to develop tBLMs: it concerns the tethering of membrane proteins, instead of the membrane lipids [175] (Figure 5). First developed by [PERSON]'s group [112], this tBLMs are called _Protein-tBLMs_ or _ptBLMs_. For this purpose, a recombinant membrane protein presenting a _'His-tag'_, e.g., a sequence of typically 6 Histidine genetically added in C- or N-terminus, and solubilized in detergent micelles is first attached by affinity to nitrilotriacetic acid (NTA) moiety grafted to a metal surface via a sulfhydryl group, in the presence of Cu\({}^{2+}\) or Ni\({}^{2+}\) cations. In a second step, the detergent molecules are substituted by lipid molecules by in situ dialysis, or in the presence of microporous biobeads and detergent-solubilized lipids, thus forming a lipid bilayer that is tethered by the protein itself. By using _cytochrome c oxidase_ (an ubiquitous membrane protein of the respiratory chain in eukaryotes) (_CcO_) as model, the successful reconstitution of the ptBLMs was assessed by SPR, QCM [112] and SEIRAS [107]. The water layer remaining interposed between the lipid bilayer and the NTA moiety can act as an ionic reservoir as demonstrated by EIS with the active protons transport across the lipid bilayer during catalytic redox cycle of the reconstituted _CcO_ in the presence of reduced cytochrome c [112]. Catalytic activity and electron transfer through functional coupling of the redox protein to the electrode (referred to as \"electronic wiring\") have been also assessed by _cyclic voltammetry_ (CV) in combination with _surface-enhanced resonance Raman spectroscopy_ (SERRS) and provides mechanistic and structural insights into the proton translocation coupled to electron transfer across the redox center, i.e., the heme center, during the catalytic redox cycle in the presence of cytochrome c under aerobic conditions [120; 240; 241] (for more details, see the review by Naumann & Knoll (2008) [132]). ptBLMs can be seen as universal systems suitable for immobilization and surface-focused reconstitution of all His-tagged membrane proteins. Purification of the proteins could even be avoided, if the preparation of ptBLMs was performed using the crude cell lysate [112]. This strategy allows controlling the orientation [122; 242] and the packing of the reconstituted membrane protein in the bilayer [243]. In contrast, it is only relevant for studies of membrane properties or protein functions in which the lateral mobility of the protein is irrelevant since all the proteins are immobilized on the surface [71]. Given examples are redox proteins, like _cytochrome c oxidase_, that one might want to connect electronically to the base electrode because they need to be \"wired\" to the support for an efficient heterogeneous electron transfer between the external circuit and the redox center of the protein [76].