Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?

Membrane proteins (MPs) are essential for cellular functions. Understanding the functions of MPs is crucial as they constitute an important class of drug targets. However, MPs are a challenging class of biomolecules to analyze because they cannot be studied outside their native environment. Their structure, function and activity are highly dependent on the local lipid environment, and these properties are compromised when the protein does not reside in the cell membrane. Mammalian cell membranes are complex and composed of different lipid species. Model membranes have been developed to provide an adequate environment to envisage MP reconstitution. Among them, tethered-Bilayer Lipid Membranes (tBLMs) appear as the best model because they allow the lipid bilayer to be decoupled from the support. Thus, they provide a sufficient aqueous space to envisage the proper accommodation of large extra-membranous domains of MPs, extending outside. Additionally, as the bilayer remains attached to tethers covalently fixed to the solid support, they can be investigated by a wide variety of surface-sensitive analytical techniques. This review provides an overview of the different approaches developed over the last two decades to achieve sophisticated tBLMs, with a more and more complex lipid composition and adapted for functional MP reconstitution

A chimiluminescent Langmuir-Blodgett membrane as the sensing layer for the reagentless monitoring of an immobilzed enzyme activity.

International audienceIn the nanotechnology field, the concept of using biomolecules as an elementary structure to develop self-assembled entities has received considerable attention. Particularly, the ability of amphiphilic molecules like lipids to self-organize into bilayers can be exploited to provide biomimetic membrane models. Langmuir–Blodgett (LB) technology, based on the transfer of an interfacial film onto a solid support, offers the possibility to prepare lipid bilayers suitable for biomolecule immobilization and achievement of nanoscale-organized sensing layers, tailored for the design of miniaturized biosensors. With the aim of immobilizing enzymes in a defined orientation at the surface of LB bilayers, an original strategy has been previously developed in our group. This approach combines two techniques based on molecular self-assembly properties: liposome fusion at an air/buffer interface and Langmuir–Blodgett technology. It allows the functional insertion of a non-inhibitory antibody in lipid bilayers, further used to anchor a soluble enzyme at the surface of the lipid membrane. When associated with an electrochemiluminescent (ECL) sensor, this molecular assembly allows the design of a biomimetic sensor able to closely integrate the recognition and transduction events. However, sensor's performance not only depends on bioactive sensing layer properties, but also on the additional introduction of luminol in the reaction medium which delays ECL reaction. This work explores the potentiality of two neosynthesized amphiphilic luminol derivatives to form a lipid bilayer serving as a matrix used for both antibody insertion and ECL detection in order to develop a new sensing layer allowing a reagentless detection. As a model, choline oxidase activity has been detected. After enzyme immobilization at the surface of the luminol derivative LB bilayer by the way of specific recognition of a non-inhibitory antibody, in situ catalytic generation of hydrogen peroxide is able to trigger ECL reaction in the sensing layer interfaced with an optoelectronic device leading to a reagentless detection of choline oxidase activity

Design of glycosyltransferase inhibitors targeting human O-GlcNAc transferase (OGT)

International audienceInhibition of glycosyltransferases requires the design of neutral inhibitors to allow cell permeation in contrast to their natural dianionic substrates. O-GlcNAc transferase (OGT) is a key enzyme involved in dynamic glycosylation of cytosolic and nuclear proteins in competition with phosphorylation. Designing OGT inhibitors is of prime interest for the better understanding of its biological implications. Introduction of a pyridine moiety as a pyrophosphate surrogate was evaluated, which provided moderate in vitro inhibition of OGT. Docking studies highlighted some key features for the binding of the designed inhibitors to the catalytic site of OGT where the carbohydrate moiety did not occupy its natural position but rather turned away and pointed to the solvent outside the catalytic pocket. Further investigation with cellular assays did not provide inhibition of OGT. This lack of OGT inhibition was rationalized with a permeation assay which revealed the sequestration of the inhibitors at the membrane

Influence of lipid packing on the membrane-binding properties of mycolactone.

<p>Change in surface pressure (Δπ, mN/m) when mycolactone interacts with mixed monolayers at different initial surface pressures (π_i, mN/m). The nature of the lipid membrane was as follows: (A) and (B) mixture 1 consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol. (C) and (D) mixture 2 consisting of 48% POPC, 41% SM, 11% POPE (given in mol%). Experiments were performed at 20°C (A) and (C), or 25°C (B) and (D). Each point corresponds to an independent measurement with a new lipid monolayer formed on PBS subphase (pH 7.4). The final concentration of mycolactone was 60 nM. Representative data from two or three independent assays are shown.</p

Influence of mycolactone interaction on the distribution of the liquid-ordered (L_o) phase in the membrane.

<p>Fluorescence images of monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol and including 0.5% BODIPY-cholesterol (TopFluor Cholesterol), after the injection (4.45 μL) of ethanol (row a) or mycolactone (row b) into the PBS subphase (pH 7.4) beneath the interfacial film compressed at an initial surface pressure of 30 mN/m at 20°C. The injection was performed after a relaxation time of one hour, and surface area was kept constant (mobile barriers were stopped). The final concentration of mycolactone was 60 nM. Scale bar: 50 μm.</p

Interaction of mycolactone with mixed monolayers in the presence or absence of cholesterol.

<p>Adsorption kinetics (π-<i>t</i>) curves of mycolactone on monolayers composed of (A) mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol) or (B) mixture 2 (48% POPC, 41% SM, 11% POPE) at 20°C (solid line) or 25°C (dashed line). Mycolactone was injected (4.45 μL) into the PBS subphase (pH 7.4) beneath the monolayer compressed at an initial surface pressure π_i of 30 mN/m after a relaxation time of one hour (arrows). Surface area was kept constant during the run. The final concentration of mycolactone was 60 nM. Each measurement was performed at least three times for each condition, and a representative curve is presented here.</p

Detergent effect on mixed monolayers in the presence of cholesterol.

<p>Adsorption kinetics (π-t) curves of detergent on monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol (mixture 1) at 20°C, and the corresponding BAM images. (A) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a final concentration of 60 nM. (B) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a constant final “Active concentration/CMC ratio” of 0.06. (C) BAM images corresponding to the adsorption kinetics (π-t) curves after the injection of Tween 20 (b) and (d), or Triton X-100 (c) and (e), at a constant final concentration of 60 nM (b) and (c), or an “Active concentration/CMC ratio” of 0.06 (d) and (e). (a) Images recorded after the injection of a 60nM mycolactone (final concentration). In all experiments, the monolayer was compressed at an initial surface pressure π_i of 30 mN/m and detergents were injected after a relaxation time of one hour (arrows). Surface area was kept constant during the run. Each measurement was performed at least three times for each condition, and a representative curve is presented here. Image scale: 483 × 383 μm².</p

Surface pressure (π)–molecular area (<i>A</i>) isotherms and corresponding BAM images of monolayers with and without cholesterol.

<p>(A) π-<i>A</i> isotherms of mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol given in mol%) recorded at 20°C (solid line) or 25°C (dashed line). (B) π-<i>A</i> isotherms of mixture 2 (48% POPC, 41% SM, 11% POPE given in mol%) recorded at 20°C (dashed-dotted line) or 25°C (dotted line). (C) Comparison of isotherms of the above-mentioned monolayers. Isotherms were recorded on PBS subphase (pH 7.4). Each isotherm corresponds to the mean of three experiments. BAM images were recorded during compression of the monolayer at a constant rate of 0.045 nm².molecule^-1.min^-1. Images A and B were recorded at 20°C. The images obtained for C were identical for 20 and 25°C. The estimated error for monolayers is ±0.05 mN/m for π and ≤ 0.01 nm² for (A). Image scale: 483 × 383 μm².</p