Different planar lipid bilayers are considered, which are designed as model systems for membrane proteins in a functionally active form. Tethered bilayer lipid membranes (tBLMs) were investigated in our group based on peptide- or oligo-oxy-ethylene (OEO) tethers. Peptide-tethered BLMs were shown to incorporate large proteins such as cytochrome c oxidase (CcO) isolated from bovine heart and F0F1 ATPase from chloroplasts, whereas OEO-tBLMs were designed to incorporate ion carriers such as valinomycin and small channel proteins such as melittin and gramicidin. The activity of these proteins could be demonstrated. However, a quantitative treatment was not feasible. Therefore, we have concentrated our efforts on a relatively new approach, the protein-tethered bilayer lipid membrane (ptBLM), which uses the protein itself as the essential building block. With the example of cytochrome c oxidase (CcO) from R. sphaeroides with His-tags attached to subunits I and II the protein was immobilized in opposite orientations and reconstituted into the respective ptBLMs. Direct electron transfer from an electrode into the enzyme could be measured by electrochemical and spectroelectrochemical methods when the enzyme was immobilized with the electron entry side directed toward the electrode. The uptake of 4 electrons per molecule CcO measured under anaerobic conditions was treated by rigorous electrochemical theory to follow a sequential, or ECCC mechanism. When the enzyme was exposed to an oxygenated solution an amplified current density indicated catalytic turnover. As far as spectro-electrochemistry was concerned, the most promising results were obtained by electrochemically controlled surface-enhanced infrared absorption spectroscopy (SEIRAS). Potentiometric titrations followed by SEIRAS indicated significant conformational changes of the protein as a function of potential. The enzyme could even be shown to adopt two different conformational states, a non-activated and an activated state before and after it was allowed to undergo catalytic turnover. Differences between the two states could be demonstrated particularly using 2D autocorrelation maps.
Mendeley saves you time finding and organizing research
Choose a citation style from the tabs below