Proteins in Tethered Bilayer Lipid Membranes

R. Naumann

Membrane proteins play a major role in every living cell. These proteins are the key factors in the cell’s metabolism, for example in cell-cell interaction, signal transduction and transport of ions and nutrients. Due to this important function, they are a preferred target for pharmaceuticals (currently more than 60% of consumed drugs). Contrary to this fundamental role in biology, functional characterization of membrane proteins remains a challenge. To address this problem biomimetic membrane systems were developed such as tethered bilayer lipid membranes (tBLMs). Proteins were shown to incorporate in a functionally active form into tBLMs in two different formats:

Thiolipid-based tBLMs were used to incorporate porines, ion carriers and channels such as hemolysin, valinomycin, melittin, gramicidin [1], MaxiK, the M2 channel of the AChR. The kinetics of ion transport through these moieties was analysed using impedance measurements as a function of various parameters, such as ion concentration, transmembrane potential, leak resistance etc. The spectra were simulated using the computer program Spice designed to analyze bioelectrochemical processes across membranes.

Larger complex proteins such as the cytochrome c oxidase from Rh. sphaeroidis, CcO, were incorporated in the so-called protein-tethered Bilayer Lipid Membrane (ptBLM) developed in our laboratories. Recombinant CcO solubilized in detergent was immobilized in different defined orientations on a chemically modified gold surface to a nickel chelating nitrilo-triacetic acid (NTA) surface. The CcO monolayer was reconstituted into the lipid by substitution of detergent molecules with lipids using in-situ dialysis or adsorption of the detergent molecules by biobeads. The kinetics of electron transfer and proton transfer was investigated by various electrochemical and surface-analytical methods including vibrational and fluorescence spectroscopy.

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