This work is aimed at establishing a quantitative physical understanding of the dynamic molecular structure of membranes using modern techniques of electron spin resonance (ESR) spectroscopy developed in the laboratory of Freed, (i.e. multi-frequency ESR and two-dimensional electron-electron double resonance [2D-ELDOR]), as well as spin-labels resembling natural components. Specific projects include the following. Studies of model membranes, wherein liquid crystalline and liquid ordered phases coexist, will provide insights into lateral heterogeneity and formation of domains in biological membranes. Studies on the plasma membranes of live RBL-2H3 mast cells and plasma membrane vesicles derived from them, will relate the observed differences in their dynamic structures to their differences in composition and molecular interactions; spectral changes resulting from cell stimulation will be related to changes in composition. The mechanistic basis for the role of lipid composition on integral membrane protein function is to be studied in membranes containing gramicidin A (GA). This includes molecular distortions on the boundary lipids that coat GA and the role of GA in lipid sorting. Measurements on spin-labeled GA will determine how the lipid and ionic membrane environments affect the equilibrium between, and structure of, GA dimer conformers. A critical assessment of gating models in the ligand-gated potassium channels KcsA and MthK will be provided. The structure of the voltage-sensing domain in KvAP will be studied. Double-quantum coherence (DQC)-ESR and DEER will be used to measure the distances in GA, KcsA, MthK and KvAP. The known fusogenicity of fusion peptides and their mutants will be correlated with changes in the ordering and motional rates they induce in the membrane lipids. Possible clinical applications include detection of membrane changes during immune response, prevention of viral entry, and neurological disorders.
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