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Membrane proteins in nanometric holes: Real-time monitoring of membrane-mediated reactions by localized surface plasmons

Final Activity Report Summary - NANOHOLES (Membrane proteins in nanometric holes: Real-time monitoring of membrane-mediated reactions by localized surface plasmons)

More than 30 % of all drugs are targeted against membrane proteins. Thus, pharmaceutical companies have a keen interest in artificial platforms that allow them to screen the function and binding interactions of isolated membrane proteins outside the cell context. However, these proteins only remain active when embedded in their natural environment, the membrane. Membranes consist of myriads of lipid molecules, which are arranged in a double layer with a water impenetrable, hydrophobic core. Because the lipids are not physically linked with each other their assemblies are very fragile and it is difficult to incorporate other water-insoluble components, such as membrane proteins, retroactively.

The key accomplishment of this project was the development of a method for the programmed ‘fusion’ of lipid assemblies. During the fusion process, all lipids and the membrane components included in the lipid assembly merged to form a uniform membrane. It was the same concept that cells used to facilitate the transport of proteins between different membrane-surrounded cell organelles. While membrane fusion was catalysed by highly specialised proteins in nature, we designed a fusion machinery from short cholesterol-modified deoxyribonucleic acid (DNA) strands. Since cholesterol was a natural membrane component with poor water-solubility, cholesterol-tethered DNA strands were spontaneously incorporated into any lipid assembly offered. This way, the surface of lipid assemblies that were intended for fusion could be activated with complementary DNA strands. After mixing those DNA-modified lipid assemblies, the complementary DNA strands formed a double helix by hybridisation. During helix formation, the lipid surfaces were pulled in close proximity and merged eventually.

We initially used this method to merge spherical lipid assemblies, i.e. vesicles, with each other and we later expanded the concept to fuse vesicles to planar lipid sheets. Planar lipid sheets were of particular interest because they are frequently used as matrix for membrane proteins in various biosensor designs. Having this tool in hands, we were able, by the time of the project completion, to build lipid architectures and embed membrane proteins at locations defined by the DNA strand used to induce fusion.