The fluidity of a membrane controls many functions vital to cell survival. Membrane fluidity depends on how tightly the lipid chains are packed which in turn is determined by the membrane components as well as the temperature and pressure.
The aims of this proposal generally fall under two categories which focus on how chain packing a) affects surface interactions to better understand how these, in turn, may change the membrane fluidity and b) the incorporation, dynamics and oligomerization of membrane proteins to understand how packing may affect protein function. The packing of model membranes will be varied by subjecting the membranes to high hydrostatic pressure at different temperatures. The use of pressure as an independent variable allows us to isolate the affects brought about by changes in packing from ones brought about by changes in the thermal energy. Model membranes are chosen because their composition can be easily varied thus allowing particular effects to be isolated. To understand how packing affects the membrane surface, changes in the stability of charged vesicles under pressure will be established by monitoring the gel to liquid crystal phase transition temperature with fluorescent probes. After, changes in the surface pH and hydrogen bonding network under pressure will be studied using a fluorescent pH indicator capable of hydrogen bonding. Finally, changes in the bilayer to hexagonal phase transition temperature will be determined to understand the effect pressure may have on lipid shape and hydration. To understand the affects chain packing may have on membrane proteins, I will first determine whether pressure changes the extent a membrane binding protein, a small integral peptide and a large integral protein incorporate into the membrane. Then, the changes in tryptophan motions of the later two proteins with pressure will be determined by the fluorescence anisotropy. Last, changes in oligomerization of dimer labelled with energy transfer pairs will be studied as a function of pressure and lipid chain structure. Through these experiments, I hope to better understand how lipid head groups and their interactions could affect membrane fluidity and how the fluidity may serve to regulate the activity of membrane proteins through changing protein incorporation, dynamics and oligomerization.
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