Upgrade to 600 MHz NMR spectrometer

Nuclear Magnetic Resonance (NMR) is a spectroscopic technique closely related to Magnetic Resonance Imaging (MRI). It is a very versatile technique. Its biggest application lies in its ability to provide detailed information on interactions between molecules. Proteins up to about 30 kDa (250 amino acid residues) can have backbone signals assigned rapidly and almost automatically. This means that we can rapidly and simply work out which signal comes from which amino acid, thereby providing a fingerprint of the protein. Addition of a ligand (for example a small molecule drug, or another protein) produces changes in signals, which are straightforwardly analysed to determine where the ligand is binding, and often to produce a binding affinity. With larger proteins, similar information can be obtained in slightly different ways although with rather more experimental effort. Very large proteins (including membrane proteins and receptors in intact cells) are still amenable to investigation, and there are straightforward techniques for identifying which ligands bind to such targets, and which parts of the ligands are most closely in contact. NMR has many other uses. It can be used to calculate the structures of proteins in solution, to analyse local and global mobility, to look at structural change on alteration of solution conditions or addition of ligands, to measure local pKa values of charged sidechains, to analyse the metabolites present in complex mixtures such as body fluids, to follow metabolic and enzymatic processes, and much more. Extracting such information is not always straightforward and often requires expert advice and intervention.
Currently we carry out many such analyses on a range of targets, often as a result of problems being investigated by external users that we are asked to advise on. Selection of projects is made on the basis of scientific importance rather than funding, although funding is clearly of considerable importance. Our current equipment is old and becoming obsolete, and is therefore likely to fail catastrophically at some point over the next few years. We are looking to replace it with new equipment, which has very similar capabilities, although the intervening years have led to improvements in many systems, for example more stable electronics and more rapid switching. The upgraded instrument will therefore be able to do the same things as our current instrument, but better and faster, with fewer instrumental artifacts and limitations. It will have some new capabilities, which will enable us to carry out a wider range of investigations, and will bring it up to the highest international standards. The current instrument has lasted 18 years: we expect the upgraded instrument to last at least another 10 and hopefully considerably longer. The funding required is thus a very cost-effective solution to the provision of world-class NMR facilities.
The upgraded instrument will be used for a wide range of projects, with users mainly from Sheffield but also further afield. These include investigation of amyloid proteins (the cause of Alzheimer's disease); fundamental research on protein stability and solubility in solution; work on the mechanism of kinases and phosphoryl transferases (key regulatory enzymes within metabolic pathways; investigations on bacterial proteins that recognise specific parts of bacterial cell walls and could therefore be used as a basis for both antibiotics and novel diagnostic tools; investigations of the function of specific proteins; and the development of high-pressure NMR as a tool for investigating the relatively low-population active forms of proteins. The instrument therefore provides an important resource for many problems in biomolecular research.

We need to upgrade our 600 MHz NMR spectrometer, which is now 18 years old. It still works well, but is gradually starting to fail, and critically is now no longer supported by the manufacturer, meaning that any failures may not be recoverable. Its age means that we are not able to implement some recent developments in NMR (specifically rapid acquisition including non-uniform sampling, temperature control of the same, and direct detection of carbon), which is an increasing frustration and is leading to some potential local users going elsewhere, with increased costs and slower progress. We have two high-field magnets, at 600 and 800 MHz. Only the 600 has a cryoprobe, meaning that the two instruments are of similar sensitivity. They are broadly similar in their specifications, and are used fairly interchangeably, so that failure of the 600 would effectively halve our throughput, which would have a very severe impact on the large number of projects that make use of it, of which a small sample is presented in the detailed case for support. We estimate that at any point, there is roughly £2.5m worth of grant funding dependent on results obtained from the 600. Thus the main argument for the upgrade is that catastrophic failure is increasingly likely, and there is a very strong business case for replacing it now: spending £450k now to remove the serious risk of delaying or losing a recurrent £2.5m makes obvious sense. The increased throughput and new functionality available with the new instrument gives us the potential to increase the user base, and thus bring in additional funding and 'enhance the capability of the UK research base in areas of science in the BSRC remit'. We propose to retain the existing magnet, and make use of existing infrastructure. Installation will be overseen by the applicants and by the Facility Manager, who will remain in post and conduct training.

There are some projects (particularly those of the applicants) where NMR results constitute the major output of the research. For these, the impact of the research is considered within those projects. There are many projects for which NMR constitutes a small (though sometimes very important) part. For example, the project described under section 2.1.6 of the case for support is a BBSRC-funded project led by Prof Kelly, investigating a novel defence-related operon in Campylobacter jejuni. He has identified the genes, obtained a structure for the main target protein Cj0424, and carried out extensive investigations of its function. He has shown that it has membrane-directed activities, but the assays are rather indirect. By NMR we were able to show that it binds specifically to lipid membranes and not to monomeric lipids, and that it binds only to negatively charged lipids. NMR has thus been able to provide a clear biophysical demonstration of the function, which was lacking from previous results. Here again the impact forms part of the project. We therefore feel that we do not need to discuss here the impact arising from individual projects that use the spectrometer.
The impact arising from this proposal is thus (we propose) a more generic impact: what NMR does and why it is useful. There are two main classes of impact to be discussed: impact on the general public, and impact on the business community.
The NMR facility is located in the basement of the Department, one floor beneath the entrance hall, departmental reception, and coffee room, and two floors beneath Firth Hall, which is a large function room used by us for open days. The building was constructed in 1905 as the main University building, and is a typical red-brick University building. The three spectrometers are housed in adjacent space, and thus present a striking appearance: three large modern pieces of scientific apparatus, housed within a traditional red-brick building. We therefore make extensive use of the NMR facility, and it is included on the itinerary of all UCAS visits, University open days, etc. This means that we have around 1500 visitors/year coming through the NMR facility. We have a range of display boards, explaining NMR, showing protein structures and ligand binding sites, and showing the construction of the Facility. On UCAS days and University open days, we have tour guides (MPW or a member of the lab) explaining how the machines are used and what they do. In addition, the Facility is on display during the two public events per year organised by the Faculty of Science. It is also used regularly to show to visitors and distinguished guests. The Vice-Chancellor's office is in the same building, and we are occasionally called on to show the Facility to official visitors to the VC's office. Once the new instrument is in place, we will upgrade our permanent displays.
We also have a display board specifically for commercial activity, in which we explain what NMR can do, with some examples. This is supported by a web page, giving further details. We shall also expand and refresh these displays. These will take place within 3 months of installation of the equipment.