S-layer lipid membranes for membrane protein reconstitution

Genome sequencing projects have revealed that membrane proteins represent about a third of the gene products in most organisms. Transmembrane (TM) proteins and membrane-associated proteins are targeted in many (infectious) diseases and thus, they are a preferred target for pharmaceuticals (currently more than 60% of all consumed drugs). Due to this important function, membrane models play an important part in unravelling the fundamental cellular processes involved and in screening for pathogens or drug candidates. In the present project lipid membranes anchored to a solid support which has previously been covered by a crystalline bacterial cell surface layer (S-layer) will be fabricated without the need of an aperture. These S-layer supported lipid membranes mimic the cell envelope structure of Archaea which dwell under very harsh conditions. Spherical, disc- and cylinder-shaped assemblies will be bound electrostatically or via specific interactions to many or few defined positions, respectively, on the S-layer lattice to form planar lipid membranes. The function of the S- layer lattice is manifold: stabilizing scaffoldings for the phospholipid bilayer and tetraetherlipid monolayer (and mixtures of the lipids), anchoring layer, spacer towards the solid support, ion reservoir, and as second layer on the top of the S-layer supported lipid membrane acting as an antifouling and nanoporous protecting layer. These S-layer supported lipid membranes differing in generation and anchoring strategy will be extensively characterized in terms of long-term stability and fluidity. Due to its planarity, a broad arsenal of techniques can be used to probe the structural and dynamic properties of these supported lipid membranes. The fluidity will be investigated by fluorescence microscopical and spectroscopical techniques. By applying the S-layer technology it is expected that the long-term robustness of fluid lipid membranes can be substantially increased. The final goal, however, is the reconstitution of functional skeletal muscle ryanodine receptor Ca2+ release channels, nicotinic acetylcholine receptors, and a-hemolysin. These TM proteins have been chosen because of their clinical importance, availability of ligands, agonists and antagonists, antibodies, representing different membrane- spanning structures, and the accumulated knowledge in the literature. Single channel recordings are envisaged on all of these TM proteins reconstituted in S-layer supported lipid membranes. This project will develop systems where TM proteins can be studied under controlled conditions not only for basic research but also in screening for ligands, pathogens or drug candidates, agonists and antagonists, and the development of TM protein-based biosensors.

Solid supported lipid membranes are versatile mimics of cell envelope structures and in particular well suited for studying biological functions like membrane-active peptides (MAPs) and (trans)membrane proteins (MPs). The novelty of the present project is the utilization of a crystalline bacterial (termed surface (S)-layer) protein as a biocompatible spacer between solid supports (e.g. microelectrodes, sensor surfaces, etc.) and lipid membranes. Whereas the primary functions of lipids are to define barrier properties and provide architectures within MAPs and MPs can reconstitute or self-assemble, the task the of S-layer lattice is to provide an anchoring structure for the lipid membrane and a tethering layer to ensure the required space and membrane fluidity which is imperatively necessary to incorporate MAPs and MPs. As membrane formation was feasible on S-layer lattices without the need of any aperture, these novel biomimetic architectures constitute an important step towards simplification and miniaturization of the whole device and are highly suitable for the investigation of reconstituted biological functions by high-resolution imaging, surface-sensitive and electro-chemical techniques.Two strategies for generation of an S-layer supported lipid membrane have successfully been developed and hence, different biologically relevant questions may be addressed. These S-layer supported lipid membranes demonstrate both, a high electrical isolation characteristics and an elevated fluidity so that MAPs and MPs can be incorporated in their functional form.MPs are very important as demonstrated by the fact that one-third of all proteins are MPs, many of them directly affected in numbers of diseases. Hence, nowadays more than 60% of all consumed drugs act on MPs such as pore-forming proteins, ion channels, receptors or enzymes. It is anticipated that in the near future the importance of MPs in medicine, pharmacology, and sensor systems (e.g. artificial nose, etc.) will increase rapidly as more and more information on its structure and function is available. This project provides not only proof of concept investigations on the incorporation and functional characterization of pore-forming proteins and receptors but allows also interesting insights in the mechanism of the insertion of (antimicrobial) MAPs in model lipid membranes. Finally, possible applications in the field of medical and technological areas like new imaging technologies may not only be applied but also refined and the obtained results may facilitate the production of high throughput screening devices for diagnostics, lead compound identification for pharmacology, and novel systems for MP-based biosensors to name just the most important ones.