The 3D structures of proteins are essential to fully characterise the sites mediating their molecular functions and their interactions with other proteins. However, whilst revolutionary technologies have enabled the sequencing of thousands of complete genomes, it is more challenging to determine the 3D structures of the proteins. Although the sequence repositories now contain >10 million protein sequences, less than 70,000 protein structures have been determined. Fortunately, in parallel with developments in sequencing technologies, powerful computational methods have emerged to predict the structure of a protein from its sequence. Currently these methods provide putative structures for ~80% of domain sequences from completed genomes, although the accuracy of this data varies from reasonably precise when structures are modelled using templates based on close relatives, through to quite approximate for models based on remote relatives and where proteins have no structurally characterised relatives. This project will bring together 6 internationally renowned UK groups involved in (1) classifying protein domains into evolutionary families (as this facilitates structure and function prediction) and/or (2) protein structure prediction. As regards the first activity - classification of protein structures - the two groups involved (SCOP,CATH) are the only groups, worldwide, providing this data. However, each applies somewhat different methodologies to make their assignments. Collaboration between these groups, in GENOME-3D, will involve comparison of domain structures and family classifications leading to refinements of assignments and/or confidence levels where the methods disagree. Since manual curation of the data is essential and since the rate at which the structures are determined is increasing, collaborations will speed up classification by allowing the groups to share information on the more challenging assignments and to discuss outcomes. For the second activity, structure prediction, the groups involved use technologies that vary in their sensitivity and in their ability to handle large numbers of sequences. Whilst SUPERFAMILY (based on SCOP) and Gene3D (based on CATH) provide greater coverage they are less likely to recognise very remote homologues, where methods such as GenTHREADER, Phyre, Fugue perform better. For each sequence, we will combine predictions from these different resources and assign confidence for each residue position in a query sequence based on the number of methods that agree in their structural prediction. We will provide pre-calculated assignments and also allow dynamic queries on the methods. We will also build 3D models for the sequences with residue positions highlighted according to agreement between the methods. We will develop computational platforms that integrate the information provided by each resource. To distribute this data to the biological and medical community we will build a dedicated web site. We will also establish web servers that link the methods ie run all the methods on query sequences and then report consensus assignments and highlight differences. In addition the consensus classification and annotation data will also be provided via two major international sites - the PDBe and InterPro. The sequence repositories are expanding at phenomenal rates as metagenomics and next gen sequencing initiatives bring in sequences from diverse microbial environments and report sequence variants occurring across different human populations or associated with different disease phenotypes. Structural data will enhance the insights available from this data. For example, known or predicted structures can reveal whether residue mutat

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Carbon dioxide (CO2) is produced by the combustion of carbon-containing molecules, such as fossil fuels, to power industry, for transportation, and in our homes. Burning fossil fuels is not only causing the level of CO2 in the atmosphere to increase (CO2 is a greenhouse gas and a major contributor to global warming) it is also depleting valuable resources that are required for the manufacture of plastics, chemicals, fertilizers - and countless other requirements of modern society. Unfortunately, CO2 is a very stable and unreactive molecule, and the only large scale process known that can remove CO2 from the atmosphere and use it to regenerate a carbon-containing fuel is biological photosynthesis (growing plants and trees). Therefore, an industrial process that could use the energy from sunlight, or a green-electricity source, to take CO2 out of the atmosphere and turn it into a useful fuel or chemical would revolutionise modern society, by supplying both our energy and material demands. Of course, no such process currently exists. The aim of this proposal is to explore a new approach for the development of such a process. Some bacteria use enzymes called formate dehydrogenases (FDHs) to catalyse the oxidation (= burning) of formate to CO2, and extract energy from this reaction in order to survive. Chemically, formate is one of the simplest hydrocarbons - it is already used as a chemical building block (feedstock) in industry, and formate 'fuel cells' are being developed. Turning a formate dehydrogenases 'in reverse' would turn CO2 into formate, a useful product. In fact, a small number of specialised bacteria use special 'tungsten-containing' FDHs to catalyse this reverse reaction - and live off the tiny amount of energy that they extract. In a pilot study we showed that the tungsten FDH from a bacterium called Syntrophobacter fumaroxidans can act as an extremely efficient electrically-driven catalyst for the reduction of CO2 to formate. In this project we aim to find out 'how the tungsten formate dehydrogenase does it'. We will start by looking for and characterising tungsten FDHs from different organisms, to find those that are the best for our experiments. Then we will apply sophisticated biochemical, electrochemical and physical techniques to aim to find out how they work - and why they work so well. Finally, we will compare our biological catalysts with available synthetic catalysts, aiming to find out how to improve the synthetic catalysts, and to develop 'demonstration devices' that show how efficient CO2 reduction catalysts can be powered by solar radiation and used in fuel cells.

The INTENSIFY project will develop new technologies that combine highthroughput screening, next-generation sequencing and microfluidics to accelerate the discovery of biomolecules with novel and desriable functions. This technology will have manifold applications ranging from biotechnology to drug discovery.

We have known for some time that daily clocks regulate rhythmical behaviour of sleep and wake in man and other animals. These daily rhythms are endogenous as they free-run in constant conditions, and do not require synchronization to external factors such as light and dark, and are therefore termed circadian ('around a day'). The major rhythm generator of the body resides within the hypothalamus of the brain, and is termed the suprachiasmatic nucleus (or SCN). The SCN has the unique properties that it will continue to oscillate when cultured in laboratory conditions. Genes regulating the circadian clock have been cloned and we know that a key feature regulating timing is how the protein products of these genes cycle in real-time around the cell. This is regulated in part by a class of enzymes called kinases, which add phosphate bonds to the protein (phosphorylation) thereby affecting its activity. One of the best known mutations of the circadian clock is a kinase (CK1e) and was discovered in hamsters over 20 years ago, causing a shortening of circadian period. This was termed the tau mutation, since the term tau is used by circadian biologists to denote period. Mutations in the same or similar kinase systems are known to induce sleep disorders in man. We have re-made this mutation in mice and shown that it accelerates behavioural activity cycles to a similar extent as hamsters. Our proposed work now aims to study how this kinase mutation accelerates the circadian clock, both in the brain and in peripheral body clocks as well. Our earlier research using hamsters has shown that the circadian clock may be accelerated by an abrupt change in phase at a specific time of day, due to accelerated turnover in the nucleus of the cell of core clock proteins. This is equivalent in mechanical terms to a gear box missing a few key cogs, causing it to jump to a new position at each rotation. We aim to test this idea in the mouse by studying protein movements in real-time using new transgenic animals which we aim to create in which key clock proteins are tagged with a fluorescent marker. These types of studies can only be addressed in mice, as these are the only animals in which it is possible to make such genetic modifications. We will use these animals to define how the kinase acts on its target proteins by studying which areas (domains) of the protein are phosphorylated by this kinase. By crossing our clock protein-tagged mice to the tau mutant animals, we will be able to define how tau accelerates period and on which proteins it acts. This is important as a description of how this is achieved could in the longer term lead to the development of novel drugs impacting on sleep and wake cycles in man. Some of core genes involved in the generation of circadian rhythms have been deleted from the genome of mice by genetic modification techniques. These so-called knock-out mice are still rhythmic as other residual clock elements are sufficient to drive behaviour. We will cross our tau mutant mice into these knock out mice and define which of the knock-outs exhibits shortening of wheel-running activity cycles. This will tell us whether tau can act in the absence of a specific gene and also the extent to which it can shorten period. By this means we aim to define which clock gene proteins are likely targets for regulation of behavioural activity cycles by tau. Finally, we aim to capitalize from the fact that the SCN and other tissues continue to oscillate in culture. We will monitor activity of cultured tissues using specialized reporters of clock genes which generate light (luciferase reporters). By monitoring light levels with specialized photon recording equipment, we will be able to examine how the circadian clock regulates timing in tissues, and their responses to stimuli which can re-set the clock.