Purchase of a 600 MHz ACAS magnet and cryogenic probe for high throughput metabolomics and ligand discovery

This proposal requests funding to purchase a sample probe and superconducting magnet to be used for a magnetic resonance spectrometry at the University of Birmingham for scientific research by a broad range of users from across the United Kingdom. This probe and magnet will be used to characterize the structures and interactions of animal, plant and microbial proteins and metabolites. This information will be used to understand the functions of these molecules in solutions similar to those in the interior of a living organism or cell. In order to obtain such information, very weak electromagnetic signals originating from the hydrogen, nitrogen and carbon atoms of the protein must be detected. The requested probe offers the highest possible level of sensitivity for these experiments, allowing researchers to observe molecules that are rare, difficult or expensive to produce, or insufficiently soluble or stable at the high concentrations required for NMR analysis. The cryogenic probe allows one to either lower the concentration of the sample or reduce the time required to obtain NMR spectra of proteins. New experiments that are otherwise precluded by sensitivity requirements can be used, allowing larger proteins and rare small molecules to be detected and data to be collected more quickly. The requested magnet offers the latest in superconducting technology at a magnetic field strength of 14 Tesla. This magnet offers excellent separation of the hydrogen, nitrogen and carbon signals, and represents the state-of-the-art standard for metabolomics and ligand discovery research. In addition this magnet is actively cooled and actively shielded, meaning that it recycles helium and does not require weekly maintenance, thus reducing manpower costs in an environmentally friendly manner. The research that would be enabled by this probe focuses on proteins and biochemical systems which are involved in regulation of cell growth, and the control of the metabolic and signalling pathways within several organisms. The organisms to be studied include food-borne pathogenic bacteria, models for genomics such as Dictyostelium, aphids and the Arabidopsis plant, zebrafish, and mammalian tissues including the human brain. The availability of this probe and magnet would be a major draw for new users, allowing more experiments to be performed, better quality data to be collected, and new projects initiated and supported within a new national facility used for internationally competitive research in the post-genomic sciences.

Cryogenic probes are essential for modern NMR spectroscopy. The primary NMR limitation is its low sensitivity due to the intrinsically small population difference of nuclear spin states. Compared to conventional probes, cryogenic probes provide a 1H and 13C sensitivity gain of 3.3 and 17 times, respectively. Consequently, it takes a fraction of the time to collect a multidimensional dataset with the same overall signal-to-noise ratio for a given sample. A cryogenic probe is often the only option to obtain the required signal-to-noise in an acceptable period of time, particularly for 3D/4D NOESY experiments which provide the majority of the restraints needed to calculate macromolecular structures. In addition, the sensitivity gain allows more users and projects to be accommodated. Higher sensitivity allows the use of new techniques which further reduce the time required to solve protein structures, including reduced dimensionality NMR and HADAMARD methods. Both advances significantly reduce the time required to record spectra but require high sensitivity. Lower protein concentrations allow larger amounts of ligands to be screened simultaneously thus increasing throughput. Some ligands are too expensive to use for such studies otherwise (e.g. labelled peptides and phospholipid analogues). Studies of rare or 13C labelled metabolites that require high sensitivity for observation of signals in biofluids, cellular milieu or extracts. The higher sensitivity of cryogenically cooled probes also allows for ligand screening of much larger 13C methyl labelled proteins using ~50 microM samples by 13C-HSQC methods. The cryogenic probe will also enable 13C-only NMR spectroscopy, which can be the only way to assign proteins having broadened NH signals due to large molecular masses, paramagnetic metal binding sites, or that require basic pH solution conditions.