We wish to modify extensively a commercial NMR spectrometer to provide an exceptionally high sensitivity proton-detected NMR spectroscopy of lower frequency nuclei, (abbreviated for convenience to HUMP, Heteronuclear Multiquantum Proton Detected NMR) particularly for 15N, 13C, and Cd, using multiquantum evolution methods in two-dimensional NMR. Modifications of frequency generation, pulse programming, and probes will be required. For 15N, we have demonstrated a three order of magnitude increase in sensitivity compared to current direct detection, and hope to obtain an additional two orders of magnitude by use of state-of-the-art electronics and improved probe designs. This will provide a completely new area of research in biomolecular structure investigation of proteins and nucleic acids, using materials with natural abundance 15N content. With 15N labeled material, HUMP can provide an unprecedently selective and sensitive probe NMR method for site specific investigation of enzyme mechanisms or effector-receptor interactions. Applications to nuclei other than 15N can potentially provide sensitivity gains, which, while lower, are nonetheless significant in combination with the two-dimensional resolution of HUMP. The development of improved indirect detection techniques will also be of general application in studies of metabolism using stable isotope labeled materials. The three year period of this proposal involves a 24-month implementation period, followed by a 12-month period of use of the instrument, on projects to which this specialized instrument can make unique contributions.
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| Live, D H; Cowburn, D; Breslow, E (1987) Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons. Biochemistry 26:6415-22 |
