Interaction of human apolipoprotein A-I with model membranes exhibiting lipid domains

ABSTRACT Several mechanisms for cell cholesterol efflux have been proposed, including membrane microsolubilization, suggesting that the existence of specific domains could enhance the transfer of lipids to apolipoproteins. In this work isothermal titration calorimetry, circular dichroism spectroscopy, and two-photon microscopy are used to study the interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) and sphingomyelin (SM), with andwithout cholesterol. Below 30C the calorimetric results show that apoA-I interaction with POPC/SM SUVs produces an exothermic reaction, characterized as nonclassical hydrophobic binding. The heat capacity change (DCp) is small and positive, whereas it was larger and negative for pure POPCbilayers, in the absence of SM. Inclusion of cholesterol in the membranes induces changes in the observed thermodynamic pattern of binding and counteracts the formation of a-helices in the protein. Above 30C the reactions are endothermic. Giant unilamellar vesicles (GUVs) of identical composition to the SUVs, and two-photon fluorescence microscopy techniques, were utilized to further characterize the interaction. Fluorescence imaging of the GUVs indicates coexistence of lipid domains under 30C. Binding experiments and Laurdan generalized-polarization measurements suggest that there is no preferential binding of the labeled apoA-I to any particular domain. Changes in the content of a-helix, binding, and fluidity data are discussed in the framework of the thermodynamic parameters