Toward Electrophoretic Separation of Membrane Proteins in Supported -Bilayers.

Membrane proteins are key constituents of the proteome of cells but are poorly characterized, mainly because they are difficult to solubilize. Proteome analysis involves separating proteins as a preliminary step toward their characterization. Currently, the most common method is "solubilizing" them with sophisticated detergent and lipid mixtures for later separation , for instance, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Filling nanopipettes with apertures smaller than 50 nm: dynamic microdistillation.

Using nanopipettes with very small apertures (<10 nm) is a good way to improve the spatial resolution in scanning conductance experiments, to monitor single-molecule delivery and to strain long molecules stretching during translocation. However, such nanopipettes can be difficult to fill. Here we describe a dynamic microdistillation technique that successfully fills all nanopipettes, whatever their shape or tip radius.

Nanoroughness Strongly Impacts Lipid Mobility in Supported Membranes.

In vivo lipid membranes interact with rough supramolecular structures such as protein clusters and fibrils. How these features whose size ranges from a few nanometers to a few tens of nanometers impact lipid and protein mobility is still being investigated. Here, we study supported phospholipid bilayers, a unique biomimetic model, deposited on etched surfaces bearing nanometric corrugations.

Electrophoretic mobility of a monotopic membrane protein inserted into the top of supported lipid bilayers.

We have studied the translational migration of a monotopic membrane protein, the bacterial sulfide quinone reductase (SQR) in supported n-bilayers ([Formula: see text]) under the influence of an electric field parallel to the membrane plane. The direction of the migration changes when the charge of the protein changes its sign. Measuring mobilities at different pH enables us to gain experimental physico-chemical data on SQR as its isoelectric point and its estimated oligomeric state (at least trimeric) when inserted in a lipid membrane.

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers.

Monotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer.

Ripple formation in unilamellar-supported lipid bilayer revealed by FRAPP.

The mechanisms of formation and conditions of the existence of the ripple phase are fundamental thermodynamic questions with practical implications for medicine and pharmaceuticals. We reveal a new case of ripple formation occurring in unilamellar-supported bilayers in water, which results solely from the bilayer/support interaction, without using lipid mixtures or specific ions. This ripple phase is detected by FRAPP using diffusion coefficient measurements as a function of temperature: a diffusivity plateau is observed.

Electric migration of α-hemolysin in supported n-bilayers: a model for transmembrane protein microelectrophoresis.

Proteome analysis involves separating proteins as a preliminary step toward their characterization. This paper reports on the translational migration of a model transmembrane protein (α-hemolysin) in supported n-bilayers (n, the number of bilayers, varies from 1 to around 500 bilayers) when an electric field parallel to the membrane plane is applied. The migration changes in direction as the charge on the protein changes its sign.

Effect of ionic strength on dynamics of supported phosphatidylcholine lipid bilayer revealed by FRAPP and Langmuir-Blodgett transfer ratios.

To determine how lipid bilayer/support interactions are affected by ionic strength, we carried out lipid diffusion coefficient measurements by fluorescence recovery after patterned photobleaching (FRAPP) and transfer ratio measurements using a Langmuir balance on supported bilayers of phosphatidylcholine lipids. The main effect of increasing ionic strength is shown to be enhanced diffusion of the lipids due to a decrease in the electrostatic interaction between the bilayer and the support. We experimentally confirm that the two main parameters governing bilayer behavior are electrostatic interaction and bilayer/support distance.

Measuring liquid meniscus velocity to determine size of nanopipette aperture.

Nanopipette aperture sizes up to 25 nm are determined here using a method based on the Poiseuille law. Pressure is applied to the backside of a liquid plug placed in the widest end of the nanopipette, resulting in an air pressure tank with an aperture at the very tip of the nanopipette. Measuring the velocity of the liquid meniscus gives the air flow and thus the aperture size.

Beyond Saffman-Delbruck approximation: a new regime for 2D diffusion of α-hemolysin complexes in supported lipid bilayer.

Cell mechanisms are actively modulated by membrane dynamics. We studied the dynamics of a first-stage biomimetic system by Fluorescence Recovery After Patterned Photobleaching. Using this simple biomimetic system, constituted by α -hemolysin from Staphylococcus aureus inserted as single heptameric pore or complexes of pores in a glass-supported DMPC bilayer, we observed true diffusion behavior, with no immobile fraction.

Diffusion of latex and DNA chains in 2D confined media.

We report a study on the dynamics of latex polystyrene beads and of DNA molecules confined in two dimensions, using fluorescence video-microscopy. We particularly focus on the character of the confined objects (hard or soft) and on the nature of the confinement: liquid (in a soap film) or solid (between two glass plates). For weak confinements, whatever the nature of confinement, we observe that DNA molecules and latex beads behave very similarly: the tighter the confinement, the slower the diffusion with a good agreement with theory.

Electrophoretic separation of large DNAs using steric confinement.

We report an alternative method for electrophoretic separation of large DNAs using steric confinement between solid walls, without gel or obstacles. The change of electrophoretic mobility vs confinement thickness is investigated using fluorescence video microscopy. We observe separation at small confinement thicknesses followed by a transition to the bulk behavior (no separation) at a thickness of about 4 mum (a few radii of gyration for the studied DNA chains).

DNA electrophoresis in dilute polymer solutions: a nonbinary mechanism.

The dynamical behavior of the neutral polymer (dextran, M(w)=2 x 10(6)) is investigated during DNA electrophoresis in a dilute solution. Using a fluorescence recovery after photobleaching setup, we measured the velocity of fluorescein-labeled dextran induced by the migration of the DNA. We found that each DNA molecule drags a large number of dextrans with it.

Simultaneous measurements of the electrophoretic mobility, diffusion coefficient and orientation of dsDNA during electrophoresis in polymer solutions.

We determined simultaneously the electrophoretic mobility, diffusion coefficient D and molecular orientation during electrophoresis of dsDNAs in polymer solutions ranging from the dilute to the semidilute regime. We established, for the first time, master scaling laws for the diffusion coefficient showing a universal behavior. A model found in the literature designed for the dilute regime allows, surprisingly, to describe the mobility data over the whole range of concentrations studied and at the same time the biased reptation with fluctuations (BRF) failed for the semidilute regime, even when constraint release of the network was taken into account.

DNA separation at a liquid-solid interface.

We demonstrate that it is possible to separate a broad band of DNA on a solid substrate without topological obstacles. The mobility was found to scale with molecular size (N) as N(-0.25), while the resolution scaled as N(0.