A macroscopic H+ and Cl- ions pump via reconstitution of EcClC membrane proteins in lipidic cubic mesophases.

Functional reconstitution of membrane proteins within lipid bilayers is crucial for understanding their biological function in living cells. While this strategy has been extensively used with liposomes, reconstitution of membrane proteins in lipidic cubic mesophases presents significant challenges related to the structural complexity of the lipid bilayer, organized on saddle-like minimal surfaces. Although reconstitution of membrane proteins in lipidic cubic mesophases plays a prominent role in membrane protein crystallization, nanotechnology, controlled drug delivery, and pathology of diseased cells, little is known about the molecular mechanism of protein reconstitution and about how transport properties of the doped mesophase mirror the original molecular gating features of the reconstituted membrane proteins. In this work we design a general strategy to demonstrate correct functional reconstitution of active and selective membrane protein transporters in lipidic mesophases, exemplified by the bacterial ClC exchanger from Escherichia coli (EcClC) as a model ion transporter. We show that its correct reconstitution in the lipidic matrix can be used to generate macroscopic proton and chloride pumps capable of selectively transporting charges over the length scale of centimeters. By further exploiting the coupled chloride/proton exchange of this membrane protein and by combining parallel or antiparallel chloride and proton gradients, we show that the doped mesophase can operate as a charge separation device relying only on the reconstituted EcClC protein and an external bias potential. These results may thus also pave the way to possible applications in supercapacitors, ion batteries, and molecular pumps.

Rheology of Ultraswollen Bicontinuous Lipidic Cubic Phases.

Rheological studies of liquid crystalline systems based on monopalmitolein and 5 or 8% of 1,2 distearoylphosphatidylglycerol are reported. Such cubic phases have been shown to possess unusually large water channels because of their ability of accommodating up to 80 wt % of water, a feature that renders these systems suitable for crystallizing membrane proteins with large extracellular domains. Their mechanical properties are supposed to be substantially different from those of traditional cubic phases. Rheological measurements were carried out on cubic phases of both Pn3 m and Ia3 d symmetries. It was verified that these ultraswollen cubic phases are less rigid than the normal cubic phases, with the Pn3 m being softer that the Ia3 d ones. Furthermore, for the Pn3 m case, the longest relaxation time is shown to decrease logarithmically with increasing surface area per unit volume, proving the critical role of the density of interfaces in establishing the macroscopic viscoelastic properties of the bicontinuous cubic phases.

Interactions of Lipidic Cubic Phase Nanoparticles with Lipid Membranes.

The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air-solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air-water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur.

Quantifying the transport properties of lipid mesophases by theoretical modelling of diffusion experiments.

Lyotropic Liquid Crystals (LLCs) are a class of lipid-based membranes with a strong potential for drug-delivery employment. The characterization and control of their transport properties is a central issue in this regard, and has recently prompted a notable volume of research on the topic. A promising experimental approach is provided by the so-called diffusion setup, where the drug molecules diffuse from a feeding chamber filled with water to a receiving one passing through a LLC. In the present work we provide a theoretical framework for the proper description of this setup, and validate it by means of targeted experiments. Due to the inhomogeneity of the system, a rich palette of different diffusion dynamics emerges from the interplay of the different time- and lengthscales thereby present. Our work paves the way to the employment of diffusion experiments to quantitatively characterize the transport properties of LLCs, and provides the basic tools for device diffusion setups with controlled kinetic properties.

Stimuli-responsive lipidic cubic phase: triggered release and sequestration of guest molecules.

New stimuli-responsive nanomaterials, made up of host-guest lipidic cubic phases (LCPs) are presented. These biocompatible, stable, transparent and water-insoluble LCPs are composed of monoolein (MO) as a neutral host, and small amounts of one of three judiciously designed and synthesized designer lipids as guest that preserve the structure and stability of LCPs, but render them specific functionalities. Efficient pH- and light-induced binding, release and sequestration of hydrophilic dyes are demonstrated. Significantly, these processes can be performed sequentially, thereby achieving both temporal and dosage control, opening up the possibility of using such LCPs as effective carriers to be used in drug delivery applications. Specifically, because of the inherent optical transparency and molecular isotropy of LCPs they can be envisaged as light-induced drug carriers in ophthalmology. The results presented here demonstrate the potential of molecular design in creating new functional materials with predicted operating mode.

Design of Light-Triggered Lyotropic Liquid Crystal Mesophases and Their Application as Molecular Switches in "On Demand" Release.

Here, we present the design and assembly of a new light-responsive functional lyotropic liquid crystal system using host-guest lipidic mesophases (LMPs). Light as an external stimulus has many advantages in comparison to other stimuli: it is milder than acids or bases, and variation of intensity and duration can provide a high level of pharmacological control. The LMPs are composed of monoolein (MO) and oleic acid (OA) as host lipids and a small amount of a judiciously synthesized lipid bearing an azobenzene photoactive unit as a guest. While preserving the structure and stability of the host lipidic aggregates, the guest lipids render them specific functionalities. Single-step and sequential light-triggered release and retention of the embedded dye molecules are demonstrated, thereby achieving exquisite temporal, spatial, and dosage control of the release, opening up the possibility of using such lipidic biomaterials as effective matrices in therapy, when a continuous release of active drugs might be toxic.

Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains.

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3-5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

Active Gating, Molecular Pumping, and Turnover Determination in Biomimetic Lipidic Cubic Mesophases with Reconstituted Membrane Proteins.

Understanding the mechanisms controlling molecular transport in bioinspired materials is a central topic in many branches of nanotechnology. In this work, we show that biomolecules of fundamental importance in biological processes, such as glucose, can be transported in an active, controlled, and selective manner across macroscopic lipidic cubic mesophases, by correctly reconstituting within them their corresponding membrane protein transporters, such as Staphylococcus epidermidis (GlcPSe). Importantly, by duly exploiting the symporter properties of GlcPSe of coupled glucose/H+ transport, the diffusion of glucose can further be tuned by independent physiological stimuli, such as parallel or antiparallel pH gradients, offering an important model to study molecular exchange processes in cellular machinery. We finally show that by measuring the transport properties of the lipidic mesophases with and without the GlcPSe membrane protein reconstituted within, it becomes possible to determine its intrinsic conductance. We generalize these findings to other membrane proteins from the antiporters family, such as the bacterial ClC exchanger from Escherichia coli (EcClC), providing a robust method for evaluating the turnover rate of the membrane proteins in general.