Formation of a Biomolecular Interactions Centre. (360G-Wellcome-062164_Z_00_Z)

Genome sequencing of the major model organisms, including man, is nearing completion, and major efforts are also underway to determine the three- dimensional structures of representatives of all of their open reading frames, essentially defining the molecular components of life itself. This is a landmark achievement, but in order to fully understand the basic mechanisms underpinning living cells and devise novel therapeutic applications, it will be necessary to identify interacting molecular partners and investigate the structures and functions of their complexes. Our application seeks to address these needs. Traditional approaches to structure-function studies, however threaten to be overwhelmed by the enormous amounts of information now being spawned by the genome projects and expected from structural genomics programmes. There is therefore a requirement to increase the efficiency with which molecular functions can be studied by both experimental and predictive approaches. We believe that this can best be achieved in the context of an integrated Centre for Biomolecular Interactions, encompassing aspects of molecular medicine, which provides a clinical focus; synthetic chemistry, which provides access to novel ligands for biological targets; and advanced biological structure-function studies. Establishment of such a Centre is the purpose of this bid. Molecular structures are the essential backdrop to all the studies we propose. The applicants are all members of the Astbury Centre for Structural Molecular Biology (ACSMB), which houses a very active and productive X-ray crystallography group, that is already equipped with the latest instrumentation. We are not, therefore, seeking additional funds for X-ray crystallography as part of this proposal. We need, however, to augment our existing facilities to enable us to investigate the structures and especially the dynamic behaviour of molecules in solution, via high-field NMR, and analyse the structures of macromolecular assemblies, which control many essential biological processes, by high-resolution EM. To date, it is still very rare for structural studies in isolation to be able to predict the details of molecular interactions, which are the basis of all important biological processes. Paradoxically, it has long been known that the affinities of such interactions are governed not only by structure but also by dynamics, since these entities are implicitly related to the thermodynamic enthalpies and entropies of binding. We propose to exploit new developments in a multidisciplinary approach to the quantitative description of key biomolecular processes using novel biophysical approaches, coupled to increased computational power, to correlate these thermodynamic parameters with structural data at atomic resolution. Our application integrates biochemists, chemists, clinicians and physicists who will address these problems via intensely collaborative approaches, which have not been possible previously. Here, we present a case for the acquisition of key instrumentation, laboratory space to house it, and the associated infrastructure support staff that will comprise a Centre for Biomolecular Interactions at the University of Leeds. A central requirement of our studies is an ability to produce and purify molecules of interest, ranging from small organic species to large macromolecules. To facilitate existing research programmes, and to allow us to develop novel technologies, many of the biological molecules of interest will be produced with non-natural functional groups or stereochemistry, or site-specific stable isotope labels. Members of the new Centre have been pioneers in applying such strategies to oligonucleotide studies and the approach will be extended to peptides, peptidomimetics and proteins. Small molecules will be produced via modern combinatorial techniques and the synergy between chemists and biochemists is therefore central to this proposal. Molecular interactions will be assessed via robotic screening, encompassing in vitro nucleic acid selections (SELEX), phage display and compound library strategies. Interactions will be quantitated using a variety of modern techniques, including ultracentrifugation, calorimetry, stopped-flow fluorescence, SELDI and surface plasmon resonance. These techniques are also applicable to the study of macromolecular folding and assembly, which is a major theme within the Centre. The binding data produced, together with detailed thermodynamic information derived from solution NMR, will be integrated with drug design and molecular modelling studies, leading to further rounds of ligand synthesis and analysis. In this way, the Centre will greatly enhance our ability to characterise interactions, understand their principle features and exploit this knowledge to develop novel therapeutic strategies, we well as highlighting the evolutionary driving forces of natural ligand partners.