Heteronuclear Multi-dimensional In-Cell NMR Spectroscopy

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DESCRIPTION (provided by applicant): Nuclear Magnetic Resonance Spectroscopy is one of the most selective methods for detailed characterization of molecular systems. Its importance in studies of biological macromolecules is well recognized particularly for structure determination and for probing molecular dynamics in purified or reconstituted systems. The use of NMR spectroscopy to characterize macromolecules in cellular environments, however, has been limited by sensitivity of detection and by the conventional approaches used for acquiring the heteronuclear multi- dimensional NMR data needed for detailed analysis. Fundamental questions regarding the properties of macromolecular components in the crowded and complex compartments contained in living cells are of prime importance to our understanding of biological function and the systems biology involved under normal and pathogenic conditions. This exploratory project is designed to exploit two new and promising NMR technologies along with advanced biosynthetic expression strategies for the purpose of overcoming past limitations and applying advanced NMR methods to study proteins and protein interactions directly in living cells and cellular environments. Cryogenically cooled probes in high field NMR spectrometers operating at 600, 800 and 900 MHz with both conventional and salt tolerant probe designs will be used to enhance the sensitivity of the experiment. Data will be acquired with reduced dimensionality experiments and analyzed by projection reconstruction methods to minimize the time needed for the multi-dimensional experiments. Fast NMR methods of this type are used to collect data on a timeframe compatible with cell viability. We envision direct observation of heteronuclear, multi-dimensional NMR spectra in living cells and their use in de novo assignment of protein resonances. These data provide the foundation for assessing global organization of proteins and monitoring protein-protein, protein-ligand, and protein-DNA interactions in vivo. The primary aim of this project is to develop powerful magnetic resonance spectroscopy (NMR) methods to characterize proteins in living cells. Understanding the behavior of individual proteins in the complex and crowded environment of the cell is critical to studies of systems biology. New fast data acquisition methods along with new generation ultra sensitive NMR probes provide enabling technologies for this in-cell spectroscopy.