SLayer Supported Lipid Membranes

3.5.1. Formation of Painted Lipid Membranes

Lipid membranes (Fig. 7A) were made from a 1% (w/w) solution of DPhyPC in n-decane (27,28). The stock solution was stored at -20°C. The orifice was prepainted with DPhyPC dissolved in chloroform (10 mg/mL) and dried with nitrogen for at least 20 min. Subsequently, the compartments were filled with the electrolyte (12 mL each). The cis cell was grounded, and the trans cell was connected by another Ag/AgCl electrode to the patch-clamp amplifier. A drop of lipid mixture was put on the Teflon brush and stroked up the orifice. Membrane formation should be seen immediately (see Note 10). Thinning of the membranes was followed by measuring the capacitance of the lipid membrane. After a constant capacitance was reached (takes approx 20-40 min), experiments to study the intrinsic parameters of the lipid membrane were performed.

3.5.2. Formation of Folded Lipid Membranes

DPhyPC was dissolved in n-hexane/ethanol (9:1). The stock solution was stored at -20°C at a concentration of 5 mg of lipid/mL. At least 30 min before the formation of the membrane, the aperture was preconditioned with a small drop of hexadecane/pentane (1:10) (Fig. 7B). Both compartments were filled to just below the aperture with electrolyte (29). A volume of 2 ^L of the lipid stock solution was spread on the aqueous surface of each compartment, and the solvent was allowed to evaporate for at least 20 min. Raising the level of the electrolyte within the compartments to above the aperture by means of the syringes led to formation of a lipid membrane, which was checked by measuring its conductance and capacitance (see Note 11).

The current response from given voltage functions was measured to provide the capacitance and conductance of the lipid membranes (30,31). A triangular voltage function (+40 to -40 mV, 20 ms) may be used to determine the capacitance of the lipid membrane. The specific capacitance is about 0.4 to 0.5 and 0.6 to 0.8 ^F/cm2 for the painted and folded membranes, respectively (9,11,32,33) (see Note 12). Membrane conductance is usually <10-8 S/cm2. The settings of the two built-in Bessel filters of the amplifier for the current monitor signal were 10 and 1.5 kHz, respectively. All experiments should be performed at room temperature (see Note 13). After each experiment, the Teflon aperture

Fig. 7. Schematic illustration of formation of (A) painted and (B) folded lipid membrane. On the lower left is an illustration of the setup (not drawn to scale), and the inset shows a drawing of the bilayer lipid membrane.

was cleaned extensively with chloroform, methanol, and ethanol and finally rinsed with Milli-Q water.

3.5.3. Recrystallization of S-Layer Proteins SbpA and SbsB

After forming the painted or folded lipid membrane, the S-layer solution was carefully injected into the trans compartment to a final protein concentration of 0.1 mg/mL. The same volume of buffer was added to the cis compartment. According to our experience, the recrystallization process of S-layer subunits on lipid membranes was generally completed within 3 h (34). If the

Fig. 8. (A) Electron micrograph of negatively stained preparation of S-layer protein SbsB isolated from G. stearothermophilus PV72 recrystallized on a monolayer made of DPhPC/hexadecylamine (molar ratio of 10:4). Bar = 100 nm. (B) Computer image reconstitution of transmission electron microscopic images of oblique S-layer lattice of SbsB. Bar = 10 nm. (Reprinted from ref. 35 with permission; © 2002, Elsevier Science.)

Fig. 8. (A) Electron micrograph of negatively stained preparation of S-layer protein SbsB isolated from G. stearothermophilus PV72 recrystallized on a monolayer made of DPhPC/hexadecylamine (molar ratio of 10:4). Bar = 100 nm. (B) Computer image reconstitution of transmission electron microscopic images of oblique S-layer lattice of SbsB. Bar = 10 nm. (Reprinted from ref. 35 with permission; © 2002, Elsevier Science.)

lipid membrane should be supported by the S-layer protein SbpA from B. sphaericus CCM 2177, 10 mM CaCl2 has to be added to the electrolyte to make recrystal-lization possible. On the other hand, no CaCl2 is needed for the recrystalliza-tion of the S-layer SbsB protein of G. stearothermophilus PV72 (Fig. 8). The closely attached S-layer lattice did not affect the specific capacitance, whereas the resistance of the membranes increased slightly (32,35-38). Recrystallization of the S-layer protein can be investigated by TEM (see Subheading 2.4.3.) on deposited negatively stained preparations or by AFM investigations (see Subheading 2.2.4.) of the lipid-coated polymer septum.

The advantages of S-layer-supported lipid membranes are the enhanced long-term stability (37,38); the stability against voltage ramps even up to 500 mV and more (39); the increased bending stiffness (40); and, hence, the higher robustness against hydrostatic pressure gradients (34,35). Thus, it might be possible to distinguish at mechanosensitive ion channels (41-43), reconstituted in S-layer-supported lipid membranes, between the curvature-induced mechanical activation and the flow-induced activation (35). In addition, the tightly attached S-layer lattice allows complete reconstitution of membrane-active peptides (37,44) but also of complex membrane proteins such as a-hemolysin (36,38,39).

4. Notes

1. For chlorination, immerse silver wire as anode in a 0.1 N HCl solution and pass a current of 10 mA for 5 min through the wire.

2. B. sphaericus CCM 2177 tends to make the medium alkaline and, thus, one has to be prepared to add H2SO4 early enough to maintain a pH of 7.2.

3. The S-layer-containing solutions should be dialyzed against large volumes, usually 3 L of distilled water, with or without CaCl2 taken at each dialysis step. Be sure to cool the distilled water to 4°C before performing dialysis.

4. Single isolated S-layer subunits from many prokaryotic organisms have shown the ability to assemble into regular lattices identical to those observed on intact cells on removal of the disrupting agents used for their isolation (e.g., on dialysis). The S-layer self-assembly processes lead to the formation of flat sheets, open-ended cylinders, or spheres. Ionic strength, temperature, protein concentration, and polymer associated with S-layers can determine both the rate and extent of assembly (for a review, see refs. 5-8).

5. Excess water has to be carefully removed before patterning in order to prevent interference fringes caused by the water film.

6. S-layer protein is completely removed by ArF (X = 193 nm) irradiation at a dose of 100 to 200 mJ/cm2.

7. Rapid filling of the micron-scale channels may be followed with an optical microscope, and capillaries may be filled even when the solution enters from both ends of the mold.

8. FITC binds to the free amino groups of the S-layer protein.

9. The amino-modified CdSe nanoparticles were prepared by an organometallic synthesis using a mixture of highly boiling primary aminoalkanes and tri-octylphosphine as the coordinating solvent. The CdSe nanocrystals from about 1 mL of freshly prepared sol were precipitated by adding a small amount of methanol. After removal of the supernatant, the particles were transferred in 5 mL of aqueous solution of 20 mM N,N-dimethyl-mercaptoethylammonium chloride and 1 mM 2-(butylamino)-ethanthiol in the case of an additional functionalization with a secondary amine. Five minutes of ultrasonic treatment led to an optically clear solution.

10. Push the Teflon brush very tightly against the septum when the lipid is stroked up the orifice. If the membrane ruptures, try it again with the Teflon brush without dropping new lipid on it.

11. If no membrane formation can be achieved, remove the lipid of the air-water interface using a suction pump, and try it again with a smaller amount of lipid. In addition, be very careful that all solutions and the electrolytes are free of any dust or other contaminants.

12. The dielectric constant for lipid membranes is taken as e = 2.1, corresponding to the average dielectric constant of a long-chain hydrocarbon.

13. If the humidity is too high or the weather is sultry, membrane formation is very rare and the membranes are usually not very stable.

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