Photosystem Two accessory proteins: structures binding sites and functions

The Photosystem Two (PSII) complex has quite rightly been described as the 'Engine of Life'. This molecular machine which is found in plants, algae and cyanobacteria is able to use the energy of sunlight to split very stable and abundant water molecules into molecular oxygen, which is released into the atmosphere as a by-product, protons which are used to generate ATP, and 'high energy' electrons which are ultimately used with the ATP to fix atmospheric carbon dioxide into sugar molecules. These carbohydrates can then be used as a fuel and to synthesise biomass for growth. Over the years there has been considerable effort to understand the structure of PSII and how it works. It is now known that PSII is made up of over 20 individual proteins, bound together as a large protein complex in a lipid membrane. Tightly associated with this complex are pigments that harvest the solar energy, small organic molecules that can transport high-energy electrons and a metal cluster comprising one calcium and 4 manganese ions. It is at this metal cluster, buried deep in the protein complex, that water binds and is oxidised to molecular oxygen, giving up its electrons. But PSII is not a perfect machine; it sometimes breaks down, especially when the sunlight is very bright, and has to be repaired. To do this the damaged PSII complex is partially disassembled into a smaller complex, and the damaged protein is recognised, specifically degraded by special proteases found within the membrane, a new protein inserted and the complex reassembled. Without this special repair mechanism PSII would be quickly inactivated in the light and plant growth and oxygen evolution would be inhibited. The purpose of our research is to understand how PSII functions, how PSII is assembled from its component parts and how it is repaired efficiently. Understanding these processes might allow us in the future to enhance photosynthesis in crop plants so that we can increase growth to help satisfy the ever increasing demand for more food and more biomass. This knowledge might also have applications in the design of new, sustainable herbicides or the design of new man-made catalysts that might act as 'artificial leaves' to provide renewable fuels from solar energy. Previous work has identified a number of small proteins that seem to be involved in the assembly, repair or optimal functioning of PSII. We propose to determine the structures of these 'accessory' proteins, so we can see how they are folded in space, and how they bind with PSII. To help do this we have made large amounts of our target proteins in a bacterium, purified the proteins and made crystals which we can use in X-ray diffraction experiments to determine the structure. Alternatively we can also use nuclear magnetic resonance (NMR) spectroscopy which has the advantage that crystals need not be made. We have also been able to make complexes between some of the accessory proteins and either the intact oxygen-evolving PSII complex or smaller protein segments. Analysis of these complexes by x-ray crystallography or NMR will allow us, for the first time, to observe how these accessory proteins engage with PSII at the molecular level. It is important to relate these structural interactions with what is happening in the cell. To do this we have made strains of a cyanobacterium that lack these accessory proteins. By studying PSII assembly and repair and PSII activity in these strains we hope to be able to identify a precise defect in PSII that can be related to the structural results. In this way we will be able learn valuable new information on how the 'Engine of Life' is assembled and maintained in a working state.

The Photosystem Two complex functions as the water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membranes of chloroplasts and cyanobacteria. The structure of dimeric PSII has been determined for the thermophilic cyanobacterium Thermosynechococcus elongatus. Not present in this structure are a number of 'accessory' proteins that are important for the assembly, repair and proper functioning of PSII in vivo. Understanding the role of these accessory proteins might provide insights into how large multisubunit membrane protein complexes are assembled and in the case of PSII might provide a way to improve PSII activity in vivo. In this application we aim to clarify the structures, binding sites and function of 6 cyanobacterial PSII 'accessory' factors with homologues in higher plants. They are Ycf48, Psb27, Psb28, Psb29, CyanoP and CyanoQ. In background work we have been able to express and isolate each of these subunits from T. elongatus in E. coli as His-tagged and non-tagged proteins and been able to obtain crystals. We have determined the structure of Ycf48 to 1.5A and CyanoP to 2.4A resolution and will determine the structures for the remaining subunits. The binding sites for Ycf48, Psb27 and Psb28 in PSII will be assessed in protein/peptide complexes or protein/PSII complexes using either NMR or x-ray spectroscopy. These results will be supported by protein cross-linking data. Potentail interactions outside PSII will be assessed by affinity chromatography approaches coupled with mass spectrometry. The physiological importance of these proteins for PSII function and biogenesis will be assessed by detailed biochemical and biophysical analysis of cyanobacterial null mutants. Overall this work will provide unprecedented structural and functional insights into the mode of operation of these accessory proteins.

1. In comparison to soluble proteins, membrane proteins are much less well-understood. There are less than unique 300 membrane protein structures. In addition, very little is known about how they are assembled. Photosystem II requires many assembly factors and chaperones for it to be synthesized in an active form. The understanding of PSII assembly, as well as being of interest in itself, should also shed light on general mechanisms of the chaperone-guided assembly of integral membrane protein complexes. 2. The global energy consumption rate at present is approaching 16 TW and will rise towards 20 TW within this decade. The energy provided by solar radiation is equivalent to 100,000 TW. That is, more solar energy strikes the surface of the earth in an hour than all the global fossil energy consumption in an entire year. About 3 billion years ago, living organisms developed molecular mechanisms to take advantage of this vast energy resource and it was their photosynthetic activity which allowed life on our planet to prosper and diversify on an enormous scale. Any improvement in our understanding of any single facet of photosynthesis may immediately have far-reaching economic implications. 3. For solar radiation to be utilized on a massive scale, while at the same time minimising the interception area and not competing with food production, efficiencies greater than that achieved by biomass production are required. In terms of solar energy conversion, the early stages of photosynthesis, including the water splitting reaction, are highly efficient, while the production of biomass is less so. For this reason it is important to understand the molecular details of the highly efficient energy conversion reactions that occur before the fixation of carbon dioxide (i.e. light reactions). 4. We believe that an early-career PDRA having gained the skills required for this project will be in a very competitive position at the end of the proposed project. Ideally, he or she will have been trained in microbiology and molecular biology, protein purification (including membrane protein purification), X-ray crystallography and NMR spectroscopy.