3-D structures of the major components of a photosynthetic membrane

Nearly all life on Earth gets the food and oxygen it needs from plants, or from simpler, bacterial photosynthetic organisms that live in oceans, lakes and ponds. These bacteria are ideal for research as they are easy to grow and study. This is essential if we are to understand the makeup of the protein-chlorophyll complexes that sit in a membrane and capture light (the LH complexes) and convert it into chemical energy (the RC complexes) that can be used to power living cells. We have already used a form of imaging called atomic force microscopy to literally 'feel' the shapes of the LH and RC protein-chlorophyll complexes in the living membrane, and to see the networks they form that act as a 'satellite dish' to harvest solar energy. We have removed these complexes from the membrane and have made 3D crystals from them, and if we could make crystals of better quality we could see the atomic details of the way that the proteins and chlorophylls harvest solar energy, transmit it between them, and then convert it to electrical energy. All these processes are extremely efficient in nature, and we have a lot to learn from them if we are to design man-made solar energy devices. This application seeks funding to make better crystals and to use them to find out how protein chlorophyll complexes harvest solar energy.

The intracytoplasmic membranes of the photosynthetic bacterium Rhodobacter sphaeroides provide a simple model for the biogenesis, spatial arrangement and function of a biological membrane. More generally R. sphaeroides brings together areas such as gene expression, membrane assembly, light harvesting, cytochrome bc1 kinetics, bacterial motility and reaction centre structure/function. Our AFM studies show how light harvesting LH2 complexes make extensive contacts for energy transfer to linear assemblies of dimeric RCLH1PufX complexes. 3D crystallization trials on the LH2 and RCLH1PufX complexes are highly encouraging and in both cases have yielded crystals that diffract to 8Å; we are seeking funding to determine the 3D structures of these complexes. A variety of approaches will be used to improve crystal quality, including the use of the crystallisation robot at Daresbury, purification of R. sphaeroides lipids and assessment of their effect on the type and quality of crystals, variation in the type of detergent used, production of complexes with altered carotenoid content, the use of mutants with altered rigidity in the LH1beta polypeptide, and production of RCLH1PufX complexes with improved thermostability. Conditions will also be found to crystallize the RCLH1PufX-(cytc2)2 supercomplex. Finally, and in parallel with these studies, the first spectroscopic studies of native and engineered RCLH1PufX complexes will be undertaken. The structure of the dimer is essential to understand quinol export, to reveal the geometry of the 56 bchl molecules that snake round the two RCs to form a single energy transfer domain, and the tendency of this complex to form the linear assemblies revealed by our AFM data. Finally, an existing array of mutants in the LH2, LH1, RC and PufX components can be examined both structurally and spectroscopically for the first time.