The functional organisation of a developing light harvesting system

Nearly all life on Earth gets the food and oxygen it needs to grow from plants or their single celled equivalents the aquatic algae. However, simpler, bacterial photosynthetic organisms live in oceans, lakes and ponds and make a significant contribution to the global food chains. These bacteria are ideal for research as they are easy to grow and study, an essential feature if we are to understand the make up of the proteins ( the tiny biological machines ) that capture light and then convert this into chemical energy that can be used to power the living cell. Our work examines a particular type of protein, a light-harvesting complex (LHC), that can capture light and pass it along a network of identical LHCs to a specialised protein, the reaction centre (RC). The RC protein converts the light energy to chemical energy in the form of a proton gradient - akin to charging up a biological battery. In order to perform their function these proteins must form ordered networks within the cells, but we do not know how this is achieved. We will use a form of imaging known as an atomic force microscope to literally 'feel' the shapes of the LHC and RC proteins as the cell makes networks of them. In this way we will follow the sequence of events that leads to the functional network inside the cells and thus unlock the secret of how nature achieves this feat of bio-engineering.

Technical Summary The proposed multidisciplinary study employs molecular genetics, biochemistry, structural biology, and state of the art physical imaging technology to study the non-covalent interactions that govern membrane protein supercomplex aggregation. Using the photosynthetic bacterium Rhodobacter sphaeroides as a model system, we will use Atomic Force Microscopy to directly analyse the organisation and topology of membrane protein complexes as the assembly reorganises to adapt to variations in light intensity and oxygen tension. Furthermore, we will exploit our previous work on engineered promoters for controlling the timing of gene expression and membrane assembly, in order to investigate the factors that influence the size and organisation of the photosynthetic membrane, which consists of hundreds of closely interacting complexes. To further examine how the individual membrane complexes interact (LH2 to LH2 and LH1 to LH2) we will also exploit our methodology for protein engineering which enables us to alter any of the complexes by changing individual residues, or more extensively, by domain swapping. Photosynthetic membranes will be gently fractionated on sucrose density gradients for AFM imaging and AFFM spectral analysis as well as fluorescence emission spectroscopy. These data will inform us as to the physical disposition of the complexes, whether they are functional for excitation energy transmission to the core RC-LH1-PufX complexes and whether the LH2 complex is added to the developing membrane serially or in parallel with the core complexes. This will not only enhance our knowledge of how photosynthetic proteins come together to generate an active PSU but will reveal the sequence of membrane protein association events which eventually must form functional networks. The individual aims are listed below. 1.To establish the range of membrane architectures available to Rb. sphaeroides WT as it adapts to oxygen tension and light intensity. 2.To use a hybrid promoter LH2 expression plasmid in a deletion strain containing only RC-LH1-PufX dimers to 'titrate' LH2 complexes into dimer-containing membranes. We want to use AFM and fluorescence spectroscopy to follow the processes by which LH2 complexes form associations with each other and with the core dimers. 3.To investigate the basis for the existence of multiple puc operons by removing the second puc operon and imaging the response of the PSU architecture in terms of intercomplex associations and energy transfer efficiency within the native membrane. 4.To use our domain swap LH2-LH1 hybrid mutants to investigate the structural determinants of functional intercomplex associations. 5.To identify the LH2 assembly factor PucC within the membrane and map its location relative to the photosynthetic apparatus. Most of these experiments will proceed concurrently as most of the deletion strains and complementation vectors have already been constructed.