Studies of the green sulphur bacterial reaction centre from Chlorobaculum tepidum

Photosynthetic organisms harvest sunlight through antenna light-harvesting complexes. Light absorbed by chromophores is transferred down an energy gradient to a reaction centre (RC) where photoinduced electron transfer occurs. A charge-separated state is generated that preserves some of the original light energy as electrochemical potential. By studying these RCs allows for us to deduce how they function through the elucidation of their structure, which ultimately allows for artificial mimics to be made. Chlorobaculum tepidum (C. tepidum) is a green sulphur photosynthetic bacterium that contains a type I RC. Light energy is transferred to the RC from chlorosomes via a soluble Fenna-Mathews-Olson (FMO) protein. Although the structure of FMO has been solved on its own, little is known about the molecular organization of the reaction centre complex. This thesis looks at two of the RC sub-units (PscB and PscD) that are water-soluble. To understand the contribution that these proteins make to RC function, they have been made in E. coli using an in-house expression vector. Using a 3C protease - iLOV - biotin acceptor domain - His10 (CLBH) tag, both PscB and PscD can be readily purified on a milligram-scale in four simple steps (Ni2+-affinity, subtractive IMAC (immobilized metal affinity chromatography) after cleavage with 3C protease, gel-filtration). PscD and PscB have been labelled with 15N and 13C for structural analysis by NMR, so far PscD has shown to partially disordered implying that a potential binding partner may be required. PscB has shown to be well structured and is in the process of having its structure elucidated. The binding PscD with FMO and ferredoxin from Arabadopsis thaliana has also been assessed by isothermal calorimetry to help identify the function of this protein. Here it is also observed that when the RC is coupled to plasmons a near 5 fold increase is observed in fluorescence enhancement as compared to RC by itself. Plasmonic metallic nanoparticles are able to drastically alter the emission of vicinal fluorophores. Metallic nanoparticles can influence the fluorescence emission of nearby molecules by enhancing the absorbance of the molecule, and by modifying the radiative decay rate of that molecule. And this is what is observed. This can further be increased when coupling the RC to the Plasmon by placing a silicon dioxide (SiO2) spacer in-between the RC and nanoparticle. This is the highest flouresence enhancement observed to date. As yet, no green sulphur bacterial RC has had its structure determined. Here, purification protocols have been developed that allow milligram quantities of a complex between the RC and FMO to be prepared. As well as identifying the best suitable detergents for solubilising and purifying the RC, two different populations of the RC have been discovered that can be separated by sucrose density gradients. Vapor diffusion, lipidic-cubic phase (LCP), bicelle, and co-crystallisation trials have been performed with pure RC-FMO. Thus far, promising crystals have been obtained when the RC has been co-crystallised with ferredoxin to 60 A. These promising crystals are the first of its type, as this is the first type 1 RC crystal obtained.