All matter consists of atoms and at the nucleus of every atom are protons and neutrons. Nuclei that contain an odd number of protons or neutrons are magnetic. Nuclear Magnetic Resonance (NMR) spectroscopy uses very powerful magnetic fields and pulses of radio waves to exploit the magnetic properties of 'odd-numbered nuclei' such as 1H (hydrogen atom or proton), 15N (nitrogen), 13C (carbon), 19F (fluorine) and 31P (phosphorus). Using this technique it is possible for scientists to identify almost every different atom in a protein, nucleic acid, or small drug molecule. This can be very useful, for example, in building up a picture of the 3-dimensional structure of one of these molecules. At Dundee University NMR spectroscopy has been used to determine the structures of nucleic acids and some proteins. The possible uses for NMR spectroscopy are much more varied, however. It is possible to determine the overall size of a protein or complex of proteins, for example, and it is possible to detect which particular atoms of a protein or other molecule undergo a change in local environment when another protein or molecule interacts with it. When proteins 'talk' to each other in the cell they very often physically interact, and NMR can therefore help scientists identify interacting partners. NMR has proven to be immensely useful in drug discovery by identifying small molecules that bind to target 'receptors' of medical interest. In this application nine scientists from the College of Life Sciences outline their ideas for using a new NMR instrument to enhance their research. There are four broad topics under investigation: 1. Bacterial protein transport 2. Nucleic acid structure and folding 3. NMR in drug development 4. Protein modification and its role in gene regulation All of these already well-funded and world-class research projects propose to use NMR spectroscopy to determine structures and/or to assess interactions between molecules. The need for an upgraded NMR spectrometer at Dundee is very great. In recent years the College has expanded its structural biology, molecular microbiology, and drug discovery programs. The machine with the largest magnet in Dundee ('11.744 Tesla' or '500 MHz' spectrometer) is 18 years old and requires upgrading. The College of Life Sciences has commissioned a central suite of smaller NMR and Magnetic Resonance Imaging (MRI) spectrometers within the new Sir James Black Centre. It is proposed here to upgrade the existing 500 MHz spectrometer (at a fraction of the cost of buying a brand new instrument) and to house it in the central 'Nuclear Magnetic Resonance Spectroscopy and Imaging Facility'. The quality of the researchers and research outlined in this proposal, coupled with the versatility, sensitivity, and cost-effectiveness of the upgraded spectrometer requested, will promote increased local, national and international collaborations between scientists, attract a constant stream of new research funding, result in high quality research publications, novel structures and pharmaceuticals, as well as teaching and training future generations of NMR experts.

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.

There are a number of experimental methods that people use to obtain answers to specific biological questions. In most cases, however, one single technique cannot provide a complete answer. Because of this, it is common practice to use more than one experimental technique and then to combine all the information together to build up a detailed picture of what is happening. One very useful techinque that is under-utilised in the UK is electron paramagnetic resonance (EPR). This method is capable of detecting free (unpaired) electrons. This is useful because the unpaired electrons act as 'spies' on the molecule and can provide information on local environment. This means that EPR can be used to report on biological (free) radicals, on (paramagnetic) metal centres in biological systems and on protein dynamics. At Leicester, we do not have access to an EPR facility that is suitable for biological work. This slows our work down because it means that we cannot obtain some of the information that we need as fast as we would like to. The current proposal seeks funding to address this problem.

Proteins are dynamic molecules. Motions within a protein molecule can be localised (e.g. bond vibrations, backbone and side chain motions) occuring on relatively fast timescales, or large scale motions (domain motions, conformational changes and slow breathing modes) that typically occur on the millisecond to second timescale. Localised fast motions can influence the chemistry in enzyme active sites; larger scale motions bring active sites together, or facilitate long range communication, for example in the transfer of electrons over large distances in redox enzymes, information transfer through signalling cascades or the folding of protein molecules. These large scale motions give rise to the concept of energy landscapes - that is the free energy surface that accommodates all conformations of the protein macromolecule that are populated. The distribution of conformational states across this landscape can be perturbed, for example by ligand or drug binding, natural variation in sequence (polymorphisms) or partner protein binding. Our knowledge of the spatial distribution and temporal exploration of these landscapes is at best limited, attributed in the main to the lack of general structural and biophysical tools to capture this information. The three dimensional structures of 'rigid' protein modules are readily accessed using conventional approaches (crystallography, NMR spectroscopy). How multiple modules communicate in complex protein systems however is not accessible using these techniques. In this application we aim to develop robust experimental methods using state-of-the-art spectroscopic, kinetic and computational methods that enable investigators to study the spatial and temporal properties of landscapes and their remodelling by small molecule/protein binding. We aim to develop these methods using mammalian nitric oxide synthases, redox enzymes that are constructed from multiple functional domain the chemistry of which is coupled to major dynamical excursions during the course of the enzyme catalysed reaction. We aim to define the structures of multiple conformational states across the landscape, define the timeconstants for their interconversion and assess the functional importance of these structural transitions in the catalytic cycle of the enzyme. By providing atomic level spatial and time resolved information on the functional dynamics in nitric oxide synthase enzymes we envisage that new opportunities will accrue to develop selective inhibitors that interfere with dynamical processes linked to function. This will reinvigorate the search for isoform specific inhibitors of these enzymes, and also provide general tools for similar analysis of other dynamic systems from which function and therapeutic intervention can be studied.

Proteins are dynamic, complex structures that facilitate cellular communication, catalysis, structure, growth and division through their interaction with other biological macromolecules, enzyme substrates and ligands. Biophysical methods are crucial for determining the structure and function of protein molecules. EPR spectroscopy has emerged as a major spectroscopic technique for the structural characterization of protein complexes, membrane protein systems, analysis of protein dynamics and the chemistry catalysed by enzymes. Modern pulsed EPR methods provide important information on protein structure through triangulation of engineered spin labels or natural 'spin active' cofactors present in proteins. EPR spectroscopy can also provide detailed electronic structural information about reactive centres (cofactors and protein based radicals) present in enzyme catalysts, and time-resolved information about the chemistry catalysed by protein systems. The contents of cells are protected and enclosed by an outer sheath or membrane composed of proteins as well as fat molecules (lipids) that form a relatively impermeable barrier. Such membranes are also found inside the cell, and form compartments that have specialised functions. The proteins found in membranes often act as gatekeepers, allowing, or sometimes actively pumping, molecules through the membrane. They also have a range of other functions such as enzymes, sensors (e.g. of hormones) and as scaffolding to provide structural support. Structural information for membrane-bound systems is scarce owing to the difficulties of applying traditional structural approaches (e.g. crystallography) to membrane systems. Spin label EPR spectroscopy provides valuable distance information from which the fold/structure of membrane proteins can be investigated. Unravelling the structure of membrane proteins is one of the major research themes in Manchester, and solid-state NMR, X-ray crystallography and cryo-electron microscopy are all employed to extract structural data. Work in this area would be significantly enhanced by the provision of EPR facilities to measure distance relationships and conformational dynamics. The Manchester group forms one node of the membrane protein structure initiative, a structural proteomics initiative sponsored by the UK research council BBSRC. RNA, in its varied forms, interacts with protein to carry out fundamental roles in the cell. Understanding the contributions of various RNAs to the control of translation in the cell forms an important theme within the structural biology and biophysics group. The understanding of molecular recognition events, including those involved in assembly of macromolecular complexes consisting of both protein and RNA are a challenge for biochemical, biophysical and structural study. The extracellular matrix group forms one of the major research centres at Manchester. The enormous size of extracellular matrix complexes, such as collagen fibrils, necessitates the use of novel structural methods. EPR spectroscopy is ideal in this regard by providing distance relationships in large protein complexes. By combining the lower resolution data from these studies, with higher resolution data for protein components, or fragments of the fibrils, a picture of the architecture of these cellular structures will emerge. Finally, catalysts in biology have properties that chemists would love to emulate. Biological reactions have exquisite specificity, even down to generating a single stereoisomer, and also do not need high temperatures and pressures. To fully understand how these processes are achieved in biology, structural biology must provide atomic structures of the protein catalysts and details (at the quantum level) of reaction mechanism. Manchester has a large grouping in this area and modern EPR facilities will provide much needed electronic structure and time-resolved information to established programmes in this area of biocatalysis.