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AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

Distillation of fermented molasses for alcohol and biofuels production uses large volumes of water. Due to environmental concern, the recycling of condensates in the fermentation medium is encouraged subsequently to a purification step. This step could be performed via reverse osmosis (RO) process, in order to remove in the retentate small organic solutes which act as fermentation inhibitors. Currently, no general rule exists to predict the selectivity of RO membranes according to the nature of organic solutes. Similarly, such insights are missing in nanofiltration processes intended to detoxify biomass hydrolysates prior fermentation. Our objective was to build and validate a molecular model of typical reverse osmosis membranes (exemples: ESPA2 and CPA2 from Hydranautics Membranes, USA), consisting in an active aromatic polyamide layer (APA) with different crosslinking rates. These models will be used in future works to develop general models of transport properties (water swelling, solute partitioning mainly) in RO membranes. The main characteristics of commercial RO membranes were retrieved from isolated APA layers (i.e. separated from the polysulfone-polyester support). Geometrical characteristics such as thickness (about 200 nm), homogeneity and swelling rates were assessed with laser ellipsometry and AFM. The composition and the reticulation rate were inferred from FTIR and XPS measurements. The sorption isotherm of water was sampled with an Intelligent Gravimetric Analyzer. The construction strategy of corresponding atomistic models of dried APA membranes via Molecular Dynamics Simulation (Discover, Accelrys, USA) is presented. The described model gives the theoretical density and neutron scattering spectra of simulated APA. These results could be compared in future works with experimental investigations

International audience; Particle formation by precipitation is an important unit operation for the production of fine solids such as catalysts, pigments, and pharmaceuticals. It is the decisive step for determining properties such as particle size, shape, and purity, which in turn determine the quality of the end product. in this study, we investigated the precipitation of BaSO(4) particles in a. hollow fiber membrane device. A solution of BaCl(2), was passed tangentially over a membrane surface and reacted with a solution of K(2)SO(4) introduced through the membrane pores. The resulting supersaturation induced nucleation and particles grew on the lumen side of the hollow fibers. Particulate fouling was observed using X-ray images with BaSO(4) as a radiocontrast agent. It was shown that this fouling was attributed primarily to fiber blockage by newly formed crystals rather than to pore blockage. Moreover, a larger inner diameter of hollow fiber was shown to slow down considerably the fouling of the device. A numerical simulation was developed to calculate the supersaturation inside a hollow fiber, and the results of the simulation were compared with experimental data.

International audience; Liquid-liquid phase membrane contactors are increasingly being used for mixing and reaction. The principle is the following: component A flows through the membrane device inlet to mix/react with component B which comes from the membrane pores. This study presents a numerical simulation using computational fluid dynamics (CFD) of momentum and mass transfer in a tubular membrane contactor for laminar flow conditions. The velocity and concentration profiles of components A-C are obtained by resolution of the Navier-Stokes and convection-diffusion equations. The numerical simulations show that mixing between A and B is obtained by diffusion along the streamlines separating both components. The mixing/reaction zone width is within the region of a few hundred of microns, and depends on the diffusion coefficients of A and B. Hollow fiber membrane devices are found to be of particular interest because their inner diameter is close to the mixing zone width.

Cell lipid membranes are the site of vital biological processes, such as motility, trafficking, and sensing, many of which involve mechanical forces. Elucidating the interplay between such bioprocesses and mechanical forces requires the use of tools that apply and measure piconewton-level forces, e.g., optical tweezers. Here, we introduce the combination of optical tweezers with free-standing lipid bilayers, which are fully accessible on both sides of the membrane. In the vicinity of the lipid bilayer, optical trapping would normally be impossible due to optical distortions caused by pockets of the solvent trapped within the membrane. We solve this by drastically reducing the size of these pockets via tuning of the solvent and flow cell material. In the resulting flow cells, lipid nanotubes are straightforwardly pushed or pulled and reach lengths above half a millimeter. Moreover, the controlled pushing of a lipid nanotube with an optically trapped bead provides an accurate and direct measurement of important mechanical properties. In particular, we measure the membrane tension of a free-standing membrane composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) to be 4.6 × 10-6 N/m. We demonstrate the potential of the platform for biophysical studies by inserting the cell-penetrating trans-activator of transcription (TAT) peptide in the lipid membrane. The interactions between the TAT peptide and the membrane are found to decrease the value of the membrane tension to 2.1 × 10-6 N/m. This method is also fully compatible with electrophysiological measurements and presents new possibilities for the study of membrane mechanics and the creation of artificial lipid tube networks of great importance in intra- and intercellular communication.