Characterizing Molecular Motion in Selected Lipid Bilayer Systems
Plasma membranes are central to the existence and function of cellular systems. These membranes are known to contain a large number of constituents and as our understanding of these complex and dynamic systems has evolved, it has become clear that even simple lipid bilayer membranes are heterogeneous, highly dynamic structures. The complexity of these structures makes studies regarding the environment inside membranes difficult. For this reason, experiments are performed using model lipid bilayers. We have produced model lipid bilayers in the form of unilamellar vesicles and have chosen a chemical probe, 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) (NBD) to incorporate into our bilayer systems. NBD is a well characterized fluorophore with strong one- and two-photon absorption bands. Two phospholipids were chosen, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), as were cholesterol and sphingomyelin in order to promote phase segregation in the model lipid bilayers. The fluorescence lifetime and anisotropy behavior of NBD in vesicles were measured using time correlated single photon counting (TCSPC). We utilized both one- and two-photon excitation to allow determination of the Cartesian components of the chromophore rotational diffusion coefficient as a function of its local environment. Fluorescence lifetime data indicated that NBD, regardless of being present as a free or tethered probe, resides in the polar region of the lipid bilayer. One- and two-photon anisotropy data have been demonstrated to provide complementary information regarding molecular motions of the chemical probe in the membrane environment and indicate changes in bilayer organization upon the addition of cholesterol and sphingomyelin.
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