Cytochrome b5 (d-b5) is an intrinsic membrane protein readily isolated from endoplasmic reticulum membranes by detergent extraction. It binds spontaneously to lipid vesicles and this resultant complex will be studied as a model membrane system. The fundamental properties of this system are now understood and we have elucidated the mechanisms of both the initial binding of the d-b5 to the membrane and the subsequent intervesicle exchange. Using the fluorescent assays for binding and exchange, the role of lipid in these two processes will be examined. The properties of the lipid vesicle will be varied by altering composition, radius of curvature, charge, and lateral homogeneity. In each case both the kinetics and the ultimate equilibrium will be monitored. The assay for d-b5 exchange uses lipid vesicles containing fluorescence quenching agents. These quenchers can be located at different depths in the bilayer and it will be possible to determine the location of the fluorescent Trp in the hydrophobic tail of the d-b5 and also the kinetics with which this topography is achieved. These fluorescence results will be confirmed by use of NMR and chemical techniques. The d-b5 complexes which are produced will be examined to elucidate the effect of the protein on the lipid bilayer. Among the properties to be monitored are the effect of the d-b5 upon: transbilayer lipid exchange (flip-flop), transbilayer lipid asymmetry, acyl chain mobility, formation of boundary lipid, and intervesicle exchange of lipid. These phenomena will undoubtably be influenced by alterations in the vesicle properties, particularly curvature. Finally, exchange of d-b5 between natural membranes will be investigated. This project will evaluate the role of intermembrane exchange of intrinsic membrane proteins in vivo. If this process does occur in vivo it will be important in membrane biogenesis and in the many desaturase reactions in which d-b5 is involved.
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