High Resolution Field Cycling NMR Spectroscopy as a Probe of Phospholipid Dynamics

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The motions and dynamics of phospholipids in membranes are important characteristics of these permeability barriers. The interfacial region, composed of the phosphate group and glycerol backbone, is perhaps the least studied, but extremely important as a contact with many proteins and other molecules that bind to or are inserted into membranes. Measuring nuclear magnetic resonance (NMR) relaxation rates for nuclei in this region of the phospholipid molecule by high resolution field cycling NMR spectroscopy provides a new way to characterize this interfacial region. The method is ideal for biological phosphates and carbonyls in ester or amide linkages where interpretation of relaxation rates is often difficult. This research will use phospholipids synthesized with 13C incorporated as a way to extend the technique to increasingly complex mixtures. A major goal will be to determine how cholesterol affects the interfacial region. The technique will also be extended to other nuclei (proton NMR field cycling) to broaden the portions of a membrane that can be analyzed. The specific biological systems studied with this novel NMR methodology will include small protein/phospholipid disks that are precursors of lipoproteins and an important enzyme in cell signaling, PTEN, whose activity is thought to be regulated by phospholipids binding to an allosteric site on the protein. The results of this research will fill in gaps in how we describe motions of phospholipids in membranes, extend the scope of this new methodology to make it more attractive and useful in other fields, and provide concrete descriptions of two important biological systems where detailed information on the interfacial behavior is not easily obtained by other means. The broader impact of this project has three components. (1) Training: Both graduate and undergraduate students (with continued inclusion of women and minorities) working on this project will be trained in NMR theory and experiments. They will also obtain experience in the preparation of biomolecules and working with model membranes. This will aid in preparing them for careers in biotechnology and pharmacology. (2) Dissemination of the results: Seminars and publication of results by the PI and students on the project will showcase the methodology. Motional parameters obtained in these studies will provide a unique data set for use as an experimental test of computational studies of bilayers. As a particular way to stimulate interest in the scientific community in using a very unique resource (the Redfield high resolution field cycler), the PI will continue to offer to run samples for interested scientists. These interactions will broaden the spectrum of uses for the technique as well as revive 31P NMR as a tool for exploring many systems. (3) Outreach outside academia: On a more general level, the unique field cycler at Brandeis will be a destination for a Boston College Women in Science and Technology field trip each year. Exposing high school women (as well as undergraduates) to different aspects of basic science, particularly showing them one-of-a-kind equipment, can improve science literacy but more importantly intensify an interest in science.