ABSTRACTCHARACTERIZATION OF MOLECULAR MOTION AND ORGANIZATION IN UNILAMELLAR LIPID VESICLESByHeather A. PillmanMembrane proteins in biological cells are involved in nearly every function of cell life. For these reasons, there is great interest in incorporating chemically selective biomolecules, such as transmembrane proteins and enzymes, into membrane-based biosensors. Proteins must be reconstituted into environments that mimic the behavior of natural plasma membranes in order for them to retain their biological specificity and activity. Synthetic lipid bilayers are compositionally simple systems that approximate the dynamic fluid nature of the cell membrane, making them attractive systems for the incorporation of biomolecules. As an initial step toward understanding bilayer systems, we have focused our research on investigating the dynamic behavior and fluidity of phospholipid vesicles lacking incorporated proteins. We have interrogated specific regions of our lipid vesicles using fluorescent "reporter" molecules to determine the dynamic behavior and molecular-scale organization of unilamellar lipid vesicles using time-resolved fluorescence spectroscopy techniques. We have used the polar chromophore nitro-2-1,3-benzoxadiazol-4-yl (NBD) tethered to the headgroups of phosphoethanolamine (PE) lipids to study the dependence of headgroup functionality on inter-lipid hydrogen bonding interactions. We gauged these interactions using a time-correlated single photon counting (TCSPC) instrument to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion dynamics in supported lipid bilayers. We have also used the untethered chromophores pyrene and perylene, imbedded in the acyl chain regions of phosphocholine (PC) lipid vesicles to determine the local viscosity and phase of the lipid bilayers as a function of bulk ethanol concentration. We have observed a structural perturbation in the acyl chain regions of the bilayer at ethanol concentrations of ca. 0.6 M, which points to the influence of controlled amounts of impurities on lipid order and phase. Lastly, we have used imbedded perylene to study the effects of excess excitation energy dissipation on the molecular-scale organization of phospholipid vesicles, and cholesterol-containing phospholipid vesicles. In both vesicle systems, we concluded that the order of the local environment of the chromophore determines the efficiency of energy transfer, and the extent of local heating. Taken collectively, the results of these experiments have provided valuable insight into the chemical and structural factors that influence lipid bilayer fluidity.
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