Characterization of Transmembrane Proteins
Characterization of the ENaC Protein fusied in Lipid Bilayer Membranes
Atomic Force Microscope (AFM) images of the epithelial sodium channel (ENaC) transmembrane proteins deposited on a lipid bilayer membrane on a porous silicon surface. Protein aggregation is noted in the pictures, individual proteins (where the arrows point) are believed to be functional proteins.
By: Dr. Khalid Tantawi
As the name suggests, lipid bilayer membranes are thin membranes with thicknesses of few nanometers of two layers of lipid molecules. The most prominent examples of lipid bilayer membranes are the plasma membrane, which isolates the biological cell from its surroundings, and the cell nucleus membrane, which marks the boundaries of the cell nucleus. There are many other cellular organelles that are equipped with membranes to separate their interior from exterior environments such as the mitochondria, Golgi apparatus and endoplasmic reticulum. Biological membranes are usually composed of phospholipids, which have hydrophilic head groups and hydrophobic fatty acid tails.
Transmembrane proteins reside in the plasma membranes of cells. Studying these proteins is of extremem importance to biologists, as they are the targets of most pharmaceutical drugs. Experiments were performed to study the epithelial sodium channel (ENaC) proteins in an artificial lipid bilayer membrane (in-vitro).
Figure 1. Contact angle measurements performed on porous silicon: A drop of water forms with a small angle on a porous silicon surface (left). The contact angle is large when a lipid bilayer membrane is formed on the porous silicon surface (middle). ENAC protein vesicles spreading in the lipid bilayer membrane (right).
Several techniques for forming supported lipid bilayers have been presented in literature, some of the commonly used techniques include the following:
Lipid vesicle spreading: this technique is the conventional method in which lipids are spread over the substrate.
Spin coating technique: this method is probably the most recent, it was studied extensively by Krapf et. al. and Simonson and Bagatolli.
Montal-Mueller method: This technique was shown to form lipid bilayers on an aperture as wide as 1.0 cm.
Painting method: This method was used to form bilayer membranes under water on an aperture with an area of 10 mm2 , but those bilayers lacked stability, and failed to incorporate large proteins in them. The painting method was used to form a lipid bilayer on an aperture as wide as 150 - 200 µm and incorporate the ENaC proteins into it.
The Langmuir-Blodgett and Langmuir-Schafer techniques: These two techniques together offer one more degree of freedom over other methods by controlling the packing density of the lipid molecules and better homogeneity over the direct spreading method. They are also more convenient to transfer a lipid bilayer on a delicate substrate as porous silicon without inducing structural damage to it than the more recent spin-coating method. Moreover height differences in the coated lipid layers have been observed in spin-coating techniques, and there would always be uncertainty of the number of bilayers being spin- coated.
The deposition of the lipid bilayer membrane was done using the Langmuir-Blodgett and Langmuir-Schaffer techniques for the advantages listed above.