Purification is a normal requirement for determining the structure of biological macromolecules. However, these structures may differ depending on whether the macromolecule is in the cell interacting with its partner, or purified from cellular components. Thus, the physiological relevance of many structures remains unknown. This project will develop new experiments to characterize biological molecules in purified form, in living cells and even in whole organisms. The technique to be used, solid-state nuclear magnetic resonance (NMR) provides information about both the structure and dynamics. An important challenge for these experiments is that they often require large amounts of material, and, therefore, the studies are often limited to simple macromolecules studied in their purified forms, rather than in a native biological context. Dynamic nuclear polarization (DNP) is a powerful technique for enhancing NMR sensitivity. This project will develop DNP experiments that capture signals from amounts of material typically found in living cells. Knowledge of structures of biological macromolecules benefits society because it enables detailed understanding of basic biological processes. The proposed activities will also benefit society through training of new scientists and outreach activities.
The project will use dynamic nuclear polarization to study full viral particles of the bacteriophage Pf1, which is both an important biological agent and a template for nanotechnology. The knowledge gained regarding the virus structure, molecular recognition, solvation, and dynamics, will contribute to a broader understanding of self-assembly. The project will also develop improvements in DNP technology. DNP operates by transferring signal strength from the unpaired electrons of stable free radicals to nuclei that are then detected in the NMR experiments. These compounds, containing two closely spaced stable free radicals, are mixed with the sample. To better deliver the radical compounds, the project will develop ways to attach them to proteins in ways that facilitate specific enhancements. Moreover, tagging the proteins will facilitate detailed studies to better understand the mechanism of polarization transfer.
