Solid state nuclear magnetic resonance (NMR) is exquisitely suited to probe atomic level structural detail of biomolecules not amendable to x-ray crystallography. However, the small nuclear magnetic moments that yield narrow resonances also result in very low inherent sensitivity in NMR. Dynamic Nuclear Polarization (DNP) is a powerful combined electron paramagnetic resonance (EPR) and NMR technique that transfers the strong polarization from unpaired electron spins to nuclear spins to boost NMR sensitivity. In our DNP experiments, we endeavor to employ frequency agile gyrotrons, and extreme cryogenic sample cooling to increase the sensitivity of solid state NMR experiments up to a factor of 20,000. This tremendous gain in sensitivity will result in acquiring data 400 million times faster than conventional NMR experiments and will have a profound impact on magnetic resonance spectroscopy and structural biology. The drastic gain in sensitivity will permit biomolecular structure determination in complex heterogeneous systems in vitro with less than a milligram of sample and in hours of experimental time, or directly in vivo in a cellular context without protein purification.
Dynamic Nuclear Polarization Instrumentation and Methodology. We are developing a novel frequency tunable gyrotron capable of changing the frequency output on a microsecond time scale. Microwave frequency agility will permit the development of advanced spectroscopic irradiation schemes on the electron spins during the DNP experiment and yield significantly more control over the DNP Hamiltonian, and much more sensitive NMR experiments. We also build our own NMR DNP probes, which can couple both high-power radio frequency and microwave power simultaneously to the sample that is spinning at the magic angle (54.7o) with respect to the magnetic field. Remarkably, the NMR probe also cools our samples to 20-90 Kelvin with cryogenic helium and nitrogen, which is required to extend the relaxation times of both the electron and nuclear magnetic moments.
In vivo NMR, Bryostatin, Protein Kinase C, and HIV/AIDS Eradication. President Obama in his state of the union address told our nation that we are within reach of eradicating HIV/AIDS. Our laboratory is invested in this tremendously important goal. Our sensitive magnetic resonance instrumentation and associated methodology will be employed to drive drug development efforts of bryostatin, a powerful activator of Protein Kinase C (PKC). Improved bryostatin analogs that can selectively target specific pathways downstream of PKC will have tremendously important implications, ranging from eradicating HIV/AIDS to new treatments to combat Alzheimer’s and cancer. For instance, bryostatin can activate latent viral reservoirs of HIV through binding to Protein Kinase C. Further development of bryostatin and similar drugs are crucial to exposing infected HIV T-cells that currently escape destruction from current anti-retroviral HIV therapy. The applications of our technology development and enhanced structure determination techniques have even wider applications to virtually every protein, molecule, and chemical architecture of structural interest to biomedical science.