Langmuir : the ACS journal of surfaces and colloids 2017 09 06() doi 10.1021/acs.langmuir.7b01636
Membrane proteins exhibiting extra- and intra-cellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered Bilayer Lipid Membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by the way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19), possessing a cysteine residue in N-terminal extremity for grafting on gold and modified in C-terminal extremity by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed to bind liposomes of variable compositions containing 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A. The formation of pep-tBLMs was characterized by Surface Plasmon Resonance imaging (SPRi), and their continuity, fluidity and homogeneity were demonstrated by Fluorescence Recovering After Photobleaching (FRAP), with a diffusion coefficient of 2.5.10-7 cm²/s, and Atomic Force Microscopy (AFM). By using variable lipid compositions including Phosphatidylcholine (PC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Phosphatidylinositol 4,5-bisphosphate (PIP2), Sphingomyelin (SM), Phosphatidic Acid (PA) and Cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by fusion of tethered proteoliposomes. AFM and FRAP characterizations allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous and continuous. The values of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2.10-7 cm²/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II) revealed that CXCR4 can be reinserted in pep-tBLMs with a functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.