Dynamics of embedded fluorophores in model bilayer lipid membranes

Membrane fluidity refers to the rate of translational, rotational, and trans-leaflet lipid diffusion in bilayer lipid membranes (BLMs). It is an important physical property of the BLM that can affect the rate at which molecules diffuse across the membrane, the signaling and communication between cells, and the activity and function of membrane proteins. Membrane fluidity often depends on the interactions between constituent lipids in the BLM or between lipids and other membrane-bound molecules or macromolecules.;This dissertation focuses on four studies that involved the use of optical techniques to measure the dynamics of fluorescently-tagged lipids in synthetic model BLM systems that mimic the behavior of cell membranes. The primary tools used for characterization were fluorescence recovery after pattern photobleaching (FRAPP), which measures translational diffusion in two-dimensional supported bilayer lipid membranes (sBLMs), and time-correlated single photon counting (TCSPC), which measures rotational diffusion in three-dimensional spherical liposomes in solution. Our results showed that the diffusion of lipid fluorophores in model BLMs can significantly increase or decrease upon the addition of molecules or macromolecules to the bilayer. For example, both sonicated and extruded 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes were less fluid upon incorporation of cholesterol, likely to the result of cholesterol molecules interacting with the hydrophobic acyl chains of the bilayer and causing the membrane to become more rigid. Addition of the lipid 1,2-dioleoyl- sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) to DOPC sBLMs again resulted in a decrease in membrane fluidity. In this case, however, the reduced fluidity likely resulted from hydrogen bonding between constituent lipid head groups in the membrane. When single acyl chain lysophospholipids were added to DOPC sBLMs, the membrane fluidity increased. This is likely due to a reduction in van der Waals interactions between hydrophobic acyl chains. However, the fluidity decreased when a fraction of the lysophospholipids was converted to fatty acids by enzymatic activity of NEST (NTE esterase domain), the catalytic domain of neuropathy target esterase (NTE). The observed decrease in fluidity is attributed to the enhanced packing of fatty acids, relative to lysophospholipids, in the hydrophobic region of the bilayer. A maximum NEST protein concentration in fluid sBLMs formed from proteoliposome reconstitution was estimated and it was demonstrated qualitatively that microsomal membrane proteins at sufficiently high concentrations can decrease the fluidity of sBLMs reconstituted from microsomes.;The results of these studies give a fundamental understanding of some of the important interactions that influence the fluidity of model BLM interfaces. The results may be useful in the design of BLM-based biosensor devices, the performance of which may depend upon BLM properties such as fluidity. They may also provide insight into the interactions that affect fluidity in cell membranes.