A novel technique is presented for engineering two dimensional, oriented, and functional protein arrays immobilized on polymer nanospheres, templated through appropriately functionalized liposomes. Cytochrome c (cyt c) adsorbs on anionic lipid bilayers via the patch of charged residues responsible for precursory complex formation during electron transfer. Cyt c arrays bound to the inside wall of large unilamellar vesicles will be immobilized through photo initiated polymerization of an encapsulated prepolymer and covalent crosslinking of the protein to the polymer matrix. Removal of the lipid bilayer will release the nanosphere and expose the functional protein surface area. The biological activity of these cyt c nanostructures will be established through a variety of methods, which will also explore their applicability as mediators in electron transfer biosensors, and as affinity probes for electron transfer proteins, and their potential to provide new insight into the macromolecular recognition processes essential for regulating biological electron transfer. The immobilization techniques presented herein can be applied to other proteins which self- assemble on lipid bilayers with specific orientation due to ligand-binding or electrostatic interactions. It is a simple, general method to obtain functional, oriented protein arrays.
