The broad objectives of the proposed research is to increase the range of biomedical systems to which high resolution NMR spectroscopy can be successfully applied. The specific goals are develop new NMR methods involving the creative use of novel radio-frequency (RF) fields to better study large bio-molecules, including the development and imoplementation of the required hardware, and the detailed applications to important current questions. The methods to be developed and refined include: the use of RF gradients to suppress solvent resonances and increase the dynamic range, the use of switched geometry RF probes to avoid radiation damping, the use of RF gradients to reduce spectral artifacts while preserving the full signal intensity, particularly of broad resonance lines, the use of RF gradient, and combinations of RF and B0 gradients to more specifically restrict the range of observed coherence pathway transformation, the use of pulsed spin locking methods to study slow motions, and to improve the sensitivity of heteronuclear cross-polarization experiments. Related to these methods are a set of hardware developments, including: Related to these methods are a set of hardware developments, including: the broad use of pin-diode switched in high resolution probes, but to switch field geometries and to increase sensitivity, the development of unique RF coil configurations for creating well defined RF field gradients. These new methods and instrumentation will be used to explore transcription activation in collaboration with Professor Gerhard Wagner of the Harvard Medical School. Pulsed spin-locking methods will be used to characterize tahe slow dynamics of GAL4 when it binds to DNA, and the N-terminal 10 kD domain of the ada protein. The DNA protein complexes suffer from low solubility, and RF gradient methods will be used to suppress the solvent signal and artifacts without introducing radiation damping. These studies will take place on both the 400 MHz spectrometer at MIT and the higher field systems at Harvard Medical School.
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