# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 1 Introduction  
Cellular membranes, and more particularly the plasma membrane, are of upmost importance in the living cells. Hosting a vast plethora of proteins, plasma membrane not only serves as a physical boundary, but also mediates exchange processes between the cell and the extracellular matrix. Cellular membranes are also essential inside the cell. They aid the different organelles to carry out their cellular functions. Furthermore, many vital biochemical processes essential for cell life are managed by the biological membranes. Only a few nanometers thick, biological membranes are very complex in terms of composition but exhibit a perfect organization at the molecular level [1]. Lipids, held together by hydrophobic interactions, play a structural role by forming a continuous self-assembled bilayer acting as a passive diffusion barrier. Proteins associated with the membrane, either transmembrane proteins or peripheral membrane proteins, respectively embedded within the lipid bilayer or transiently associated with it, represent \(\sim\)30% of the open reading frames in complex organisms [2]. Due to their abundance, they are the key factors of the cell metabolism, involving cell-cell communication, cell adhesion, nutrient import, signal transduction, biocatalysis processes, energy production and others [3]. As a result, membrane receptors are currently the target of over 60% of medicinal drugs [4; 5]. Nowadays, cell membranes are no longer considered as a simple double lipid layer but as a set of complex and dynamic protein-lipid structures and segregated microdomains, that serve as functional spatiotemporal platforms for the interaction of lipids and proteins involved in cellular signaling pathways [6; 7; 8; 9; 10]. The membrane composition, and thereforethe overall function of the cell membranes, is altered in a wide range of human diseases, including cancer, neurodegenerative disorders, cardiovascular pathologies, obesity, etc. A lipid alteration can affect the localization and activity of transmembrane proteins and thus impact on the intracellular cell signaling. From this belief, a new concept of membrane lipid therapy (MLT) has emerged [11; 12] with the idea that lipid components of biological membranes can also be selectively targeted to induce membrane disorder and reverse the malfunction [13]. This approach now represents a target of choice for pharmaceutical companies [14]. Hence, investigating membranes and membrane proteins (MPs), including lipid-lipid, protein-lipid or ligand-protein receptor interactions, is of critical importance. However, due to their complexity, in situ investigations to unlock the secret of the biological membranes remains a great challenge. In this respect, the development of artificial models that mimic the cell membrane by constitution and composition, is an asset to study biological membrane properties. In the plasma membrane, hundreds of different lipid species can be found. For instance, some of them have a negative charge, which can promote interactions with positively charged amino acids in proteins [11]. Depending on the size of their polar head group, certain lipids allow docking of bulky protein lipid anchors or form tightly packed areas to help some membrane proteins to bind to regions where these lipids are abundant. It is now well-accepted that the membrane lipid composition may have a profound role in membrane functioning and cell signaling [13]. In this respect, the crucial role of non-bilayer lipids present in large amounts in biological membranes on the MP activities must be underlined [15; 16]. Conversely, reconstitution of functional membrane proteins after in vitro production or purification is challenging. Due to their amphiphilic nature, they are prone to early denaturation during in vitro handling. To properly evaluate their functionality, they require a native lipid environment.