Lipidic-sponge phase crystallisation and time-resolved Laue diffraction studies of bacterial photosynthetic reaction centres

Photosynthesis is the procedure of light capture and converting it into a chemical energy source for the cell. The work-horse of this process are photosynthetic reaction centres, which capture photons with the help of their antenna complexes and use these photons to drive charge separation reactions, and ultimately chemiosmotic coupling. It is twenty five years since the first structure of the bacterial photosynthetic reaction centre from Blastochloris viridis was solved, which also earned the Noble prize in chemistry. In this thesis we aimed to perform time-resolved Laue diffraction studies of photosynthetic reaction centres. Numerous technical challenges presented themselves. First and foremost, excellent quality crystals were essential. Thus, we investigated the crystallisation process of the Rhodobacter sphaeroides bacterial reaction centre and discovered that these crystals grew from a lipidic-sponge phase, rather than a lipidic cubic phase as thought. From here we developed a screen for lipidic-sponge phase crystallisation potentially applicable to any membrane protein. The major advantage of this novel crystallisation technique is the implementation of a liquid lipid phase that allows the application of a hanging-drop vapour diffusion experiment. Crystals of the reaction centre from Bl. viridis thus obtained were optimised, and a structure to 1.95 Å resolution determined. From this structure we observed an ancient memory of a putative enzymatic activity in ubiquinone biosynthesis. Finally, time-resolved Laue diffraction experiments on both the R. sphaeroides and Bl. viridis crystals were tried, with the latter being more successful. We observed that 3 ms following photo-activation at room temperature, a tyrosin (Tyr162) of Bl. viridis undergoes a 1.05 Å movement towards the special pair. This result is interpreted chemically, and its implication for the evolution of oxygen evolving reaction centres discussed