LABELED SYNTHETIC DPGS AND NOVEL CROSS-LINKERS FOR EM

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Two areas form the focus of this proposal. The first is an extension of our membrane protein labeling studies and has as its objective the development for the first time of chemically well defined, labeled, unsymmetrically substituted diphosphatidylglycerols of immediate importance to biochemical and biophysical studies of lipid-protein interactions in biological membranes. The study of these interactions is an important segment of the long range goal of understanding at the molecular level how cells function. The new synthetic DPGs will be used in collaborative studies with Drs. Griffith and Jost to determine the DPG binding site(s) on cytochrome oxidase through photolabeling experiments, and, through incorporation of a proxyl nitroxide spin label, to determine the number of binding sites and the relative equilibrium constants for each class of binding sites. The possibility of extending solid phase synthesis techniques to the synthesis of complex lipids will be explored during the course of this work. Complementary to the study of biological membranes and other cellular components by biophysical techniques has been the direct visualization of cellular ultrastructure through electron microscopy. Chemical fixation is the most widely used method of preservation of biological samples for transmission EM. Saturated lipids and carbohydrates in particular are difficult to fix (i.e. hold in place) using existing reagents. The second area of this proposal has as its objective the development of new cross-linking agents specifically designed to fix these important cellular constituents. These new agents incorporate combinations of the following reactive groups: p-nitrophenylboronic acid for carbohydrate binding, photoreactive carbene precursors for non-specific C-H insertions into saturated lipids and other residues, and a glutaraldehyde group that should mimic the reactivity of the widely used fixative agent glutaraldehyde.