Toward Electrophoretic Separation of Membrane Proteins in Supported n-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 via, for instance, sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, this later step induces loss of 3D structure (denaturation). Migration in a medium that mimics the cell membrane should therefore be more appropriate. Here, we present a successful electrophoretic separation of a mixture first of two and then of three different membrane objects in supported n-bilayers. These "objects" are composed of membrane proteins sulfide quinone reductase and α-hemolysin. Sulfide quinone reductase forms an object from three monomers together and self-inserts into the upper leaflet. α-Hemolysin inserts as a spanning heptamer into a bilayer or can build stable dimers of α-hemolysin heptamers under certain conditions. By appropriately adjusting the pH, it proved possible to move them in different ways. This work holds promise for separating membrane proteins without losing their 3D structure, thus their bioactivity, within a lipidic environment that is closer to physiological conditions and for building drug/diagnostic platforms.

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. Even elongated or bent nanopipettes with a small-angle tip are completely filled using this new technique. The nanopipettes are first filled with pure water, which is later replaced with the desired electrolyte via electromigration. Electrical measurements are used to check that filling is complete.

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. The surface roughness and mean curvature are carefully characterized by AFM imaging using ultrasharp tips. Neutron specular reflectivity supplements this surface characterization and indicates that the bilayers follow the large-scale corrugations of the substrate. We measure the lateral mobility of lipids in both the fluid and gel phases by fluorescence recovery after patterned photobleaching. Although the mobility is independent of the roughness in the gel phase, it exhibits a 5-fold decrease in the fluid phase when the roughness increases from 0.2 to 10 nm. These results are interpreted with a two-phase model allowing for a strong decrease in the lipid mobility in highly curved or defect-induced gel-like nanoscale regions. This suggests a strong link between membrane curvature and fluidity, which is a key property for various cell functions such as signaling and adhesion.

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. Consequently, in addition to the migration study of membrane proteins in a lipid environment, this experimental system, previously used with a transmembrane protein, is thus suitable to define membrane protein properties in conditions approaching the native ones (in the absence of detergent).

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. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological system.

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. It occurs in the same temperature range where ripple phase existence has been observed using other methods. When AFM experiments are performed in the appropriate temperature range the ripple phase is confirmed.

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. Its electrophoretic mobility is shown to depend on size and charge. The electrophoretic mobility varies as 1/R(2), with R the equivalent geometric radius of the embedded part of the protein. Measuring mobilities at differing pH in our system enables us to determine the pI and the charge of the protein. Establishing all these variations points to the feasibility of electrophoretic transport of a charged object in this medium and is a first step toward electrophoretic separation of membrane proteins in n-bilayer systems.

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. Both these parameters can therefore be used to vary the potential that acts on the bilayer. Additionally, our findings show that FRAPP is an extremely sensitive tool to study interaction effects: here, variations in diffusion coefficient as well as the presence or absence of leaflet decoupling.

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. Aperture determinations are in good agreement with SEM estimations and the proposed method is simple, relatively fast, and cheap.

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. We find two situations: i) when incubation is shorter than 15 hours, the protein inserts as a heptameric pore and diffuses roughly three times more slowly than its host lipid bilayer; ii) incubation longer than 15 hours leads to the formation of larger complexes which diffuse more slowly. Our results indicate that, while the Saffman-Delbruck model adequately describes the diffusion coefficient D for small radii, D of the objects decreases as 1/R(2) for the size range explored in this study. Additionally, in the presence of inserted proteins, the gel-to-fluid transition of the supported bilayer as well as a temperature shift in the gel-to-fluid transition are observed.

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. For strong confinements between solid walls (thickness of confinement smaller than the bulk radius of gyration), DNA coils are not immobilized and still diffuse. We show in this case that the conformation of DNA chains is in good agreement with the predictions of De Gennes and Brochard (radius approximately e (-1/4), with e the confinement gap); on the other hand, we cannot really check the theoretical predictions for the diffusion coefficient. Interestingly, strong confinement of latex beads in a soap film leads to a anomalous slow diffusion, certainly associated with an additional viscous drag generated by the interfaces.

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). We present tentative explanations of our original observations.

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. We show that DNA-dextran interactions are not only binary but long range and indirect. We conclude that the DNA-dextran complex creates a hydrodynamic field that entrains polymers far from the DNA during electrophoresis.

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(0.75) indicating that diffusivity on this substrate was minimal. By varying the buffer concentration we were able to show that the mobility for a given chain length scaled with the persistent length (p) as p(1/2). This could be shown to be related to the Gaussian conformation of the chains adsorbed on the surface. A two-dimensional corrugated surface of nonporous silica beads was produced using a self-assembling process at the air/water interface. Even though the surface corrugations were comparable to persistence length we show that they do not affect the mobility, indicating that surface friction rather than topological constraints are the predominant mechanism of separation on a surface.

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. These quantitative determinations of D are of practical interest to evaluate band broadening during capillary electrophoresis and provide data for stimulating investigation of the physics of DNA electrophoretic motion.