RESUMO
A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.
Assuntos
Bicamadas Lipídicas/química , Fosfolipídeos/química , Análise Serial de Proteínas , Receptores CXCR4/química , Ouro , Membranas , Proteolipídeos/química , Ressonância de Plasmônio de SuperfícieRESUMO
Membrane proteins exhibiting extra- and intracellular 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 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 the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 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 protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery 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 the 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 the fusion of tethered proteoliposomes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value 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 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.
Assuntos
Fosfolipídeos/química , Bicamadas Lipídicas , Fosfatidilcolinas , Receptores Acoplados a Proteínas GRESUMO
The chemokine CCR5 receptor is target of maraviroc, a negative allosteric modulator of CCR5 that blocks the HIV protein gp120 from associating with the receptor, thereby inhibiting virus cellular entry. As noted with other G-protein-coupled receptor family members, the role of the lipid environment in CCR5 signaling remains obscure and very modestly investigated. Controversial literature on the impact of cholesterol (Chol) depletion in HIV infection and CCR5 signaling, including the hypothesis that Chol depletion could inhibit HIV infection, lead us to focus on the understanding of Chol impact in the first stages of receptor activation. To address this aim, the approach chosen was to employ reconstituted model lipid systems of controlled lipid composition containing CCR5 from two distinct expression systems: Pichia pastoris and cell-free expression. The characterization of receptor/ligand interaction in terms of total binding or competition binding assays was independently performed by plasmon waveguide resonance and fluorescence anisotropy, respectively. Maraviroc, a potent receptor antagonist, was the ligand investigated. Additionally, coarse-grained molecular dynamics simulation was employed to investigate Chol impact in the receptor-conformational flexibility and dynamics. Results obtained with receptor produced by different expression systems and using different biophysical approaches clearly demonstrate a considerable impact of Chol in the binding affinity of maraviroc to the receptor and receptor-conformational dynamics. Chol considerably decreases maraviroc binding affinity to the CCR5 receptor. The mechanisms by which this effect occurs seem to involve the adoption of distinct receptor-conformational states with restrained structural dynamics and helical motions in the presence of Chol.