Studies of the interaction of myristoylated proteins, Visinin-Like Proteins, with biomimetic membranes and conception of a new membrane model dedicated to protein / lipid interaction studies

Deux membres des Visinin-Like Proteins (VILIPs), VILIP-1 et VILIP-3, ont été étudiés à l'aide de deux modèles membranaires biomimétiques, les monocouches de Langmuir couplées à la microscopie à l'angle de Brewster (BAM) et les bicouches lipidiques supportées (SLB) visualisées par microscopie à force atomique (AFM). A l'aide de ces deux modèles, nous avons pu montrer que les VILIPs, protéines N-myristoylées et possédant quatre mains-EF, ont une cinétique d'interaction membranaire qui augmente en présence de calcium, probablement dû à la présence d'un mécanisme type « switch calcium-myristoyle ». En revanche, l'utilisation de protéines mutées, non myristoylées, a révélé que la présence du groupement myristoyle n'est pas le seul facteur nécessaire pour que ces protéines interagissent avec la membrane. La présence d'une région N-terminale riche en résidus lysine permettrait à cette famille de protéines d'interagir via des interactions électrostatiques avec des membranes possédant des lipides anioniques et plus particulièrement du phosphatidylinositol-4,5-biphosphate (PIP2). La présence d'un faible pourcentage de ce phosphoinositide dans la membrane est responsable de l'accélération de la vitesse d'interaction membranaire des VILIPs, ce qui est cohérent avec leur location subcellulaire in cellulo. Enfin, un nouveau modèle membranaire de bicouches lipidiques suspendues sur des pilotis peptidiques (pep-tBLM) greffés sur une surface d'or a été ensuite développé. La méthode présentée dans ce manuscrit permet de créer des tBLM, de la composition lipidique souhaitée, en utilisant un peptide pilotis spécifiquement conçu durant cette thèse. La création de ce modèle a été suivie en temps réel par imagerie de résonance plasmonique de surface (SPRi) et caractérisé par AFM et par microscopie de fluorescenceTwo members of the Visinin-Like Proteins (VILIPs) family, VILIP-1 and VILIP-3, have been studied using two biomimetic membrane models, the Langmuir monolayers coupled to the Brewster angle microscopy (BAM) and the supported lipid bilayers (SLB) visualized by atomic force microscopy (AFM). Using these two models, we have shown that VILIPs, N-myristoylated proteins with four EF-hands, have a membrane interaction kinetic that increases in the presence of calcium, probably due to the presence of a "calcium-myristoyl switch" mechanism. Tn contrast, the use of unmyristoylated proteins revealed that the presence of the myristoyl group is not the only factor necessary for the interaction of these proteins with the membrane. The presence of a N- terminal lysine-rich region allows this family of proteins to interact through electrostatic interactions with membranes containing anionic lipids and particularly the phosphatidylionisitol-4,5-biphosphate (PIP2). The presence of a small percent of phosphoinositide in the membrane is responsible for the acceleration of the binding rate of VILIPs, which is consistent with their subcellular location in cellulo. Finally, a new membrane model of peptide tethered lipid bilayers (pep-tBLM) grafted onto a gold surface was developed. The method described in this manuscript allows the formation of tBLM, containing the desired lipid composition, by using a home-designed peptide as tether. The formation is followed in real time by surface plasmon resonance imaging (SPRi) and has been characterized by AFM and fluorescence microscop

Tethered Bilayer lipid membranes (tBLMs) : interest and applications for biological membrane investigations.

International audienceBiological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes

Micro-contact printing of PEM thin films : effect of line tension and surface energies

International audiencePolyelectrolyte multilayer (PEM) thin films are popular candidates for surface coating due to their versatility, tunability and simple production method. Often these films are used in a 2D structured manner for creating defined cell scaffolds or electronic applications. Although these films were successfully printed in the past, the conditions and energies necessary for a successful printing were only investigated as isolated parameters or as a function of the substrate but not the PEM surface energy and therefore the dominating forces remained controversial. We hereby present a theory and method for microcontact printing of condensed polyelectrolyte multilayer thin films, based on surface energies and the line tension. The theory relies on the surface energy of the substrate, stamp and PEM as well as the PEM line tension ratios to create the desired pattern. The presented theory is able to predict the printability, quality and resolution limit of a chosen system and was evaluated with experiments. A reduction of the production time from the beginning of PEM assembly to the final pattern from several hours down to 30 minutes was achieved while increasing reproducibility and resolution of the printed patterns at the same time. We would like to point out that this approach can generally be used for any kind of adsorbed thin film on substrates

Comparison of VILIP-1 and VILIP-3 binding to phospholipid monolayers

International audienceThe neuronal calcium sensor proteins Visinin-like Proteins 1 (VILIP-1) and 3 (VILIP-3) are effectors of guanylyl cyclase and acetyl choline receptors, and transduce calcium signals in the brain. The “calcium-myristoyl” switch, which involves a post-translationally added myristoyl moiety and calcium binding, is thought to regulate their membrane binding capacity and therefore, play a critical role in their mechanism of action. In the present study, we investigated the effect of membrane composition and solvent conditions on the membrane binding mechanisms of both VILIPs using lipid monolayers at the air/buffer interface. Results based on comparison of the adsorption kinetics of the myristoylated and non-myristoylated proteins confirm the pivotal role of calcium and the exposed myristol moiety for sustaining the membrane-bound state of both VILIPs. However, we also observed binding of both VILIP proteins in the absence of calcium and/or myristoyl conjugation. We propose a two-stage membrane binding mechanism for VILIP-1 and VILIP-3 whereby the proteins are initially attracted to the membrane surface by electrostatic interactions and possibly by specific interactions with highly negatively charged lipids head groups. The extrusion of the conjugated myristoyl group, and the subsequent anchoring in the membrane constitutes the second stage of the binding mechanism, and ensures the sustained membrane-bound form of these proteins

Specific interaction to PIP2 increase the kinetic rate of membrane binding of VILIPs, a subfamily of Neuronal Calcium sensors (NCS) proteins.

International audienceVIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family

Specific interaction to PIP2 increase the kinetic rate of membrane binding of VILIPs, a subfamily of Neuronal Calcium sensors (NCS) proteins.

International audienceVIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family

New Tethered Phospholipid Bilayers Integrating Functional G‑Protein-Coupled Receptor Membrane Proteins

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-<i>sn</i>-glycero-3-[(<i>N</i>-(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

Adsorption of VILIPs to monolayers of different lipid compositions under varying buffer conditions.

<p>Histograms of averaged Δ∏ for three different lipid monolayers in the absence or presence of calcium: myr-VILIP-1 (blue), VILIP-1 (red), myr-VILIP-3 (green), and VILIP-3 (purple). The phospholipid monolayers are composed of A) DMPS/DMPC (at molar ratio 3:1), B) DOPS/DOPC (3:1), and C) DOPS/DOPC (1:3) as indicated. The subphase consisted of 10 mM Hepes at pH 7.4, 150 mM NaCl, 2 mM CaCl₂ or 2 mM EDTA. Conditions in the presence or absence of calcium are shown in full and striped color, respectively. The error bars indicate the standard deviation of between two and five technical repeats.</p

Adsorption of myristoylated VILIPs and BSA to phospholipid monolayers.

<p>Surface pressure (Δ∏) change vs. time after injection of myr-VILIP-1, myr-VILIP-3 or Bovine Serum Albumine (BSA) into subphase beneath phospholipid monolayers of DOPS/DOPC (at molar ratio 3:1) compressed at an initial surface pressure (∏i) of about 16 mN/m. The final concentration of myr-VILIP-1 (red,○?), myr-VILIP-3 (blue, ⋄), and BSA (black, □) was 30 nM. The subphase consisted of 10 mM HEPES at pH 7.4, 150 mM NaCl, 2 mM CaCl2. After proteins injection (indicated by an arrow), the surface pressure increased until saturation at the equilibrium adsorption pressure (∏_e). Note that the dotted line is the tangent to the curve at initial time of protein injection and corresponds to the initial rate of binding to lipid monolayer.</p

Analysis of binding parameters of myristoylated VILIPs.

<p><b>A.</b> Surface pressure changes (Δ∏) vs. initial surface pressure (∏_i) of myr-VILIP-1 (• and ○) and myr-VILIP-3 (♦ and ⋄) in the presence of calcium (• and ♦) or EDTA (○ and ⋄). MIP values were obtained by extrapolating respectively the curves to the x axis. <b>B.</b> Equilibrium adsorption surface pressure (∏_e) vs. initial surface pressure (∏_i) of myr-VILIP-1 (• and ○) and myr-VILIP-3 (♦ and ⋄) in the presence of calcium (• and ♦) and EDTA (○ and ⋄). The phospholipid monolayers are composed of DOPS/DOPC (at molar ratio 1:3) and compressed at different initial surface pressures. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093948#pone.0093948.s002" target="_blank">Table S1</a>.</p