Lipid bilayers supported

Protems can be physisorbed or covalently attached to mica. Another method is to innnobilise and orient them by specific binding to receptor-fiinctionalized planar lipid bilayers supported on the mica sheets [15]. These surfaces are then brought into contact in an aqueous electrolyte solution, while the pH and the ionic strength are varied. Corresponding variations in the force-versus-distance curve allow conclusions about protein confomiation and interaction to be drawn [99]. The local electrostatic potential of protein-covered surfaces can hence be detemiined with an accuracy of 5 mV. 

The fact that the anions of the uncouplers are large, often aromatic, and therefore soluble in the lipid bilayer supports this interpretation the protonated uncouplers can diffuse into the mitochondria and the anion can diffuse back out. Mitochondria can also be uncoupled by a combination of ionophores, e.g., a mixture of valinomycin (Fig. 8-22), which carries K+ into the mitochondria, plus nigericin, which catalyzes an exchange of K+ (out) for H+ (in).172... 

Other biomolecules that are of interest in p.CP are lipids and lipid bilayers. Supported lipid bilayers are very fragile assemblies that are formed by lipids that are organized into two opposing leaflets on hydrophilic surfaces, such as glass or mica substrates. These structures can be also patterned on solid substrates but the p.CP technique differs slightly from the ones that were applied for proteins or DNA. First, the bilayer has to be formed on the oxidized PDMS stamp from the buffer solution by lipid vesicle fusion. Second, printing has to be carried out in water, otherwise the bilayer will lose its structure.99 This method allows efficient and reliable transfer of membrane patches to glass surfaces. 

Fig. 8 shows an AFM image of a DPPC bilayer formed using a combination of the LB technique and the Langmuir-Schaefer (LS) method [18]. In this approach, the first monolayer is deposited onto the substrate using the LB dipping technique. The second monolayer is deposited on the first using the LS approach where the substrate is positioned parallel with the air-water interface and transferred through the interface. This results in a Y-type lipid bilayer supported on a substrate. 

A new class of artificial membranes, based on lipid bilayers supported on porous alumina, were recently studied by Hennesthal and Steinem using AFM [58], In this case, tip-sample convolution effects affecting the detection of pores (see Section 2.3) were clearly demonstrated by the authors on comparing the average pore size of the alumina support as obtained by SEM (60 nm) and by AFM (50 nm). 

Exposure of octadecanethiol monolayer to the small phospholipid vesicles leads to their unrolling and adsorption of a single lipid layer on top of the octadecanethiol film. Unrolling of vesicles on the surface of the hydrophilic monolayer, however, leads to the formation of the lipid bilayer on top of the original film. This principle was used by Evans and coworkers to create lipid bilayers supported by cholesterol moieties present in a mixed gold-thiol monolayer (Figure 28)430. 

Ehospholipid Bilaver Membranes. A lipid bilayer supported on an aperture between the LAPS and the controlling electrode can be represented by adding the equivalent circuit for a lipid bilayer (5) in series with the LAPS, as in Figure 3. As a thin insulating film, the membrane has a resistance (Rm), and a capacitance (Cm) associated with it. In addition, ion gradients across the membrane may cause trans-membrane potentials. These potentials are represented by the voltage source v in the equivalent circuit. It is assumed that no low resistance pathways around the membrane are present. We would like to be able to measure Rm, Cm, and Vm. 

Gaede, H.C., Luckett, K.M., Polozov, I.V., and Gawrisch, K. 2004. Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores. Langmuir 20 7711-7719. 

Solid supported bilayer (SSB) A single lipid bilayer supported on top of a flat solid surface, such as freshly cleaned silica or a polymeric surface. 

As lipids, we used DOPC, DOPS, and DOPE (5 2 3 weight ratio). The lipid bilayer supported on a mica surface was prepared by the same method described in section 19.5.1. Two-dimensional crystallization of annexin V on the lipid bilayer was performed by injecting an annexin V solution into the observation buffer containing 50 mM Tris-HCl pH 8.0, 5 mM KCl, 2.5 mM MgC, and 3mM CaCl2. 

Supported lipid bilayers on planar silicon substrates have been formed using S-layer protein from B. coagulans E38/vl and from B. sphaericus CCM 2177 as support onto which l,2-dimyristoyl-OT-glycero-3-phosphocholine (DMPC) (pure or mixtures with 30% cholesterol) or DPPC bilayers were deposited by the Langmuir-Blodgett-technique (Pig. 

In reconstitution experiments, the self-assembly of the pore-forming protein a-hemolysin of Staphylococcus aureus (aHL) [181-183] was examined in plain and S-layer-supported lipid bilayers. Staphylococcal aHL formed lytic pores when added to the lipid-exposed side of the DPhPC bilayer with or without an attached S-layer from B coagulans E38/vl. The assembly of aHL pores was slower at S-layer-supported compared to unsupported folded membranes. No assembly could be detected upon adding aHL monomers to the S-layer face of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice did not allow passage of aHL monomers through the S-layer pores to the lipid bilayer [142]. 

We have proposed that vesicle aggregation is probably related to the disposition of pardaxin bound in the phosphatidylserine vesicle lipid bilayer (26). This conclusion is supported by the observation that phosphatidycholine vesicles are not induced to aggregate and that the pardaxin-induced phosphatidylserine vesicle aggregation is affected by charge polarization of the vesicle (26). This suggestion seems to be consistent also with the voltage dependence of fast "pore" activity of pardaxin, the channels which are open only at positive membrane potentials. 

Although for the moment this model is only partially supported by experimental data it offers the opportunity to design new experiments which will help to understand the mechanisms of pardaxin insertion and pore formation in lipid bilayers and biological membranes which at a molecular level are the events leading to shark repellency and toxicity of this marine toxin. 

The antiparallel strand structure between residues 131 and 238 in the cytoplasmic portion of Ca -ATPase was originally designated as transduction domain the name suggested its possible role in the conformational coupling between the nucleotide binding and phosphorylation sites exposed to the cytoplasm and the Ca channel located at some distance from each other in the lipid bilayer [8,42]. The site specific mutagenesis of conserved amino acids in the P strand sector of the molecule provides support for its proposed function in conformational transitions [103,126,127,215]. 

The artificial lipid bilayer is often prepared via the vesicle-fusion method [8]. In the vesicle fusion process, immersing a solid substrate in a vesicle dispersion solution induces adsorption and rupture of the vesicles on the substrate, which yields a planar and continuous lipid bilayer structure (Figure 13.1) [9]. The Langmuir-Blodgett transfer process is also a useful method [10]. These artificial lipid bilayers can support various biomolecules [11-16]. However, we have to take care because some transmembrane proteins incorporated in these artificial lipid bilayers interact directly with the substrate surface due to a lack of sufficient space between the bilayer and the substrate. This alters the native properties of the proteins and prohibits free diffusion in the lipid bilayer [17[. To avoid this undesirable situation, polymer-supported bilayers [7, 18, 19] or tethered bilayers [20, 21] are used. 

Richter, R. P., Berat, R. and Brisson, A. R. (2006) Formation of solid-supported lipid bilayers an integrated view. Langmuir, 22, 3497-3505. 

Majd, S. and Mayer, M. (2005) Hydrogel stamping of arrays of supported lipid bilayers with various lipid compositions for the screening of drug-membrane and protein-membrane interactions. Angew. Chem. Int. Ed., 44, 6697-6700. 

Munro, J. C. and Frank, C. W. (2004) Insitu formation and characterization of poly (ethylene glycol)-supported lipid bilayers on gold surfaces. Langmuir, 20, 10567-10575. 

Kam, L. and Boxer, S. G. (2003) Spatially selective manipulation of supported lipid bilayers by laminar fiow steps toward biomembrane microfiuidics. Langmuir,... 

Several works have been reported for macroscopically orientated biological membranes.106-109 The biomembrane alignment can be carried out mechanically or magnetically. The first one relies on the deposition of lipid bilayers on the surface of a rigid support (glass plates) such that the bilayer normal is perpendicular to the surface of the support itself. Small peptides and the lipid bilayers can be dissolved in organic solvents which are successively removed under vacuum.105 The re-hydration of the system in a chamber of an optimized temperature, humidity and time gives rise to the desired orientation. 

Here, we discuss a solid-state 19F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of 19F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes... 

Fig. 2 Mechanically oriented bilayer samples as a membrane model for ssNMR. (a) Illustration of the hydrated lipid bilayers with MAPs embedded, the glass supports, and the insulating wrapping, (b) A real sample consists of 15 stacked glass slides, (c) Schematic solid-state 19F-NMR lineshapes from an oriented CF3-labelled peptide (red), and the corresponding powder lineshape from a non-oriented sample (grey), (d) Illustration of typical orientational defects in real samples - the sources of powder contribution in the spectra...

For the orientation-based structure analysis of MAPs, uniformly oriented lipid bilayers are typically prepared on solid supports as illustrated in Fig. 2 [23, 47, 55]. These mechanically oriented membranes are advantageous for static ssNMR experiments, as they provide a robust way to orient a sample with any desired lipid composition, peptide concentration, and at any desired temperature. The lipids... 

Fig. 5 Membrane models for NMR structure analysis, (a) An isotropic detergent micelle (left) is compared to the dimensions of lipid bilayers (right), (b) Macroscopically oriented membrane samples can be prepared on solid support, as nanodiscs, or as magnetically oriented bicelles. (c) Nomenclature and variability of liposomes small (SUV, 20-40 nm), intermediate (IUV, 40-60 nm), large (LUV, 100-400 nm), and giant unilamellar vesicles (GUV, 1 pm) multi-lamellar (MLV), oligo-lamellar (OLV) and highly heterogeneous multi-oligo-lamellar vesicles (MOLV)...

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