Beyond Freezing: Low Temperature Lipidic Cubic Phase As Biomimetic, Nanoconfining Matrix

Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to drug delivery. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, the development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. A family of synthetic lipids with designed cyclopropyl modifications in the hydrophobic chains was synthesised in order to study the relationship between lipid molecular structure and mesophase behaviour. These lipids show a unique liquid-crystalline behaviour at low temperatures, enabling the use of LCP crystallisation in conditions never explored before.1 Please click Additional Files below to see the full abstract

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or Nycodenz-adjusted density of the hydrated detergent micelle11Dedication: In memory of our late friend Martin Zulauf, who crashed with his ‘Ultralight’ on June 17, 1995 in France. He published more than 50 papers on detergents and their interactions with proteins. It was always a pleasure to work with him and we hope that this modest contribution will be in his sense.

AbstractThe determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within ±15% to those reported previously, indicating a general applicability of this approach

Many-body Landau-Zener dynamics in coupled 1D Bose liquids

The Landau-Zener model of a quantum mechanical two-level system driven with a linearly time dependent detuning has served over decades as a textbook paradigm of quantum dynamics. In their seminal work [L. D. Landau, Physik. Z. Sowjet. 2, 46 (1932); C. Zener, Proc. Royal Soc. London 137, 696 (1932)], Landau and Zener derived a non-perturbative prediction for the transition probability between two states, which often serves as a reference point for the analysis of more complex systems. A particularly intriguing question is whether that framework can be extended to describe many-body quantum dynamics. Here we report an experimental and theoretical study of a system of ultracold atoms, offering a direct many-body generalization of the Landau-Zener problem. In a system of pairwise tunnel-coupled 1D Bose liquids we show how tuning the correlations of the 1D gases, the tunnel coupling between the tubes and the inter-tube interactions strongly modify the original Landau-Zener picture. The results are explained using a mean-field description of the inter-tube condensate wave-function, coupled to the low-energy phonons of the 1D Bose liquid.Comment: 13 pages, 10 figures

Membrane Bound Enzyme Hosted in Liquid Crystalline Cubic Phase for Sensing and Fuel Cells

Monoolein or phytantriol liquid crystalline cubic phase and carbon nanotubes were employed as the matrices for the immobilization of a membrane bound enzyme–fructose dehydrogenase (FDH). Entrapment within the cubic phase protects the guest protein from chemical and physical degradation, thereby facilitating retention of its native conformation and bioactivity. The mesophase environment was therefore found appropriate for retaining FDH close to the electrode surface. Phytantriol was used as the cubic phase component in case of measurements carried out in biological fluids containing hydrolyzing enzymes. Fructose dehydrogenase shows direct electron transfer when embedded in the matrix, thus does not require any mediators. The bioanode was employed as a biosensing device for fructose. Due to the resistance of fructose dehydrogenase to oxygen it was found useful for the construction of a membrane less biofuel cell. The enzymatic fuel cell based on FDH in the cubic phase film at the anode and laccase at the biocathode showed open circuit potential of 703 ± 10 mV in the presence of 40 mM fructose in buffer solution, and power density of 850 μW cm−2 at 250 mV in solutions under conditions of continuous flow of dioxygen

The targeted anti-oxidant MitoQ causes mitochondrial swelling and depolarization in kidney tissue

Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain

Tailored Host-Guest Lipidic Cubic Phases: A Protocell Model Exhibiting Nucleic Acid Recognition

A classical conundrum in origin-of-life studies relates to the nature of the first chemical system: was it a carrier of genetic information or a facilitator of cellular compartmentalization? Here we present a system composed of tailor-made nucleolipids and hydrated monoolein, which assemble at ambient temperatures to form host–guest lipidic cubic phase (LCP) materials that are stable in bulk water and can perform both functions. As such, they may represent a molecular model for a protocell in origin-of-life studies. Nucleolipids within the lipidic material sequester and bind selectively complementary oligonucleotide sequences from solution by virtue of base-pairing; noncomplementary sequences diffuse freely between the LCP material and the bulk aqueous environment. Sequence specific enrichment of nucleic acids within the LCP material demonstrates an effective mechanism for selection of genetic material in these cell-mimetic systems

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

Overcoming Endocytosis Deficiency by Cubosome Nanocarriers

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule