Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

M. Alejandra Tricerri, Juan D. Toledo, Susana A. Sanchez, Theodore L. Hazlett, Enrico Gratton, Ana Jonas, and Horacio A. Garda

Department of Biochemistry, Universidad Nacional de La Plata, La Plata, Buenos Aires 1900

Corresponding Author: aletricerri{at}yahoo.com

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains has been suggested to trigger cholesterol and phospholipid efflux and transport. We analyzed here apoA-I interaction with bilayers containing dimyristoyl and distearoyl phosphatidylcholine (DMPC/DSPC), at a temperature where they show phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on Giant Unilamellar Vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphtalene (Laurdan). A decrease of vesicles size was detected as long as the mixed liposomes were incubated with lipid-free apoA-I, together with a deformation of the original round shape and a relative enrichment of the target GUV in the solid component (DSPC). Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the General Polarization of Laurdan. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which the protein removes lipids, at a molar ratio similar to the domains compositions. To further characterize the products of the interaction, apoA-I was incubated with DMPC/DSPC Small Unilamellar Vesicles, and products were isolated and quantified. Protein solubilized both lipids, but formed complexes relatively enriched in the liquid component. We also show changes in the GUVs morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs at particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.

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