This work is aimed at establishing a quantitative physical understanding of membrane dynamics in model and biological systems using modern techniques of electron spin resonance (ESR) Spectroscopy, developed in the aboratory of Freed, and spin-labels closely resembling natural components. Specific projects include the following. Two-dimensional electron-electron double resonance (2D-ELDOR) and far-infrared (FIR)-ESR will be employed to better characterize the boundary lipids surrounding the peptide gramicidin A (GA), and to study the process of HII phase formation as GA is added. These modern techniques will also be used to characterize the nature and size of fluid and gel domains in two phase systems, including the effects of GA. The influence of the peripheral protein cytochrome c on the membrane will be studied by modern ESR methods. Rat basophilic leukemia (RBL-2H3) cell plasma membranes will be studied to determine if aggregation of IgE receptors at the cell surface alters the membrane dynamic structure. This will build on initial ESR and 2D-ELDOR studies wherein use of a headgroup labeled sphingomyelin yielded substantial distinctions between stimulated and unstimulated cell lysates, and where detergent-resistant membrane domains were found to have a liquid ordered phase component. The changes in dynamic structure occurring during phospholipase D activation and its membrane remodelling will be studied in model membranes resembling Golgi membranes. The (sub)-microsecond time resolution of 2D-ESR will be utilized for time-resolved studies of light-induced changes in the distance between specifically-labeled sites in bacteriorhodopsin to provide insight into the conformational changes associated with transient channel opening. This research is relevant to studies of membrane morphology, and would benefit disciplines where it is important to probe the physical response of membranes. Possible clinical applications include the study of transmembrane drug delivery mechanisms, detection of membrane changes during immune response, and diagnosis of membrane disorders in diseases of the blood, lungs, liver and kidney.
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