Membrane-mimetic systems : Novel methods and results from studies of respiratory enzymes

The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described. In the first two studies, described in Papers I & II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis. The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III). Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV).At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.