Final Activity Report Summary - ORGANIZATION (Spatial organization of photosynthetic complexes in membranes) The project aims at studying the underlying mechanisms for the organisation of photosynthetic complexes in a membrane. In most photosynthetic purple bacteria, the photosynthetic apparatus consists of the Light-harvesting complexes 2 (LH2) and Light-harvesting complex 1 (LH1), which is closely associated with the photosynthetic reaction centre. In order to elucidate, which parameters are crucial for the self-organisation of the components of the photosynthetic unit in a membrane, we study the photosynthetic complexes in model membrane systems. Model membrane systems offer the advantage that parameters as the membrane composition as well as the protein concentration in the membrane can sensitively be controlled. This way, the diffusion and supramolecular assembly of the complexes can be studied as a function of different parameters, which might be crucial for an efficient organisation of the complexes in a membrane. As a model membrane system, giant unilamellar vesicles (GUV) are used. The size of GUVs (> 10 µm) significantly exceeds the optical diffraction limit and therefore allows for studying the reconstituted, autofluorescent protein complexes by fluorescence wide-field imaging. The first step in the preparation of GUVs with incorporated photosynthetic complexes comprises the reconstitution of the light-harvesting complexes into preformed large unilamellar vesicles (r ~ 70 nm) resulting in the formation of proteoliposomes. The reconstituted light-harvesting complexes were spectroscopically characterised on the ensemble and single-molecule level. In the next step, the proteoliposomes were used for the preparation of GUVs. The GUV preparation requires the formation a dry and rather homogeneous proteoliposome film. This proteoliposome film is subsequently rehydrated and subjected to an alternating electric field (electroformation technique), which was shown to facilitate the GUV formation. GUVs with a relatively high amount of LH complexes incorporated into the GUV membrane could be prepared. Formation of the vesicles and incorporation of the photosynthetic complexes into the GUV membrane was verified by fluorescence wide-field imaging. In the following step, we will employ fluorescence wide-field imaging to study the diffusion and supramolecular assembly of the light-harvesting complexes. This will be done for various conditions in order to access information about which parameters influence the membrane organisation of the photosynthetic complexes.
