Organization and dynamics of the proteolipid complexes formed by annexin Vand lipids in planar supported lipid bilayers

The consequences of the binding of annexin V on its lateral mobility and th at of Lipids were investigated by means of experimental and simulated FRAP experiments. Experiments were carried out on planar supported bilayers (PC/ PS 9:2 mol/mol mixtures) in the presence of 1 mM CaCl2 in the subphase. The probes C-12-NBD-PS and fluorescein-labeled annexin V were used and the dat a compared with that previously obtained for C-12-NBD-PC [Saurel, O., Cezan ne, L., Milon, A., Tocanne, J. F., & Demange, P. (1998) Biochemistry 37, 14 03-1410]. At complete coverage of the lipid bilayer by the protein (C-annex in = 80 nM), the lateral mobility of C-12-NBD-PC was reduced by 40% while C -12-NBD-PS and bound annexin V molecules were nearly immobilized (D < 10(-1 1) cm(2)/s). At moderate protein concentration (20 nM < C-annexin < 80 nM), best fitting of the lipid and protein probe recoveries was achieved with o ne single diffusion coefficient and a mobile fraction close to 100%, indica ting homogeneous lipid and protein populations. In contrast, at low protein concentration (C-annexin < 20 nM), C-12-NBD-PS showed a two-component diff usion. The slow PS population at C-annexin < 20 nM and the single PS popula tion at C-annexin > 20 nM moved at the same rate that bound annexin V (mobi le fraction close to 100%), indicating strong PS/protein interactions. With the aid of computer simulations of the lateral motion of PC molecules, bas ed on the 2-D crystalline networks formed by annexin V in contact with the lipid bilayer, these FRAP results may be accounted for by considering a rat her simple model of a proteolipidic complex consisting of an extended 2-D c rystalline protein network facing the lipid bilayer and stabilized by stron g interactions between annexin V and PS molecules. In this model, immobiliz ation of annexin V and PS molecules originates from their mutual interactio ns. The slowing down of PC molecules is due to various obstacles to their l ateral diffusion which can be described as: the four PS molecules bound to the protein, the tryptophan 187 which presumably interacts with the lipids at the level of their polar headgroups and probably the three other hydroph obic amino acid residues located on the AB calcium-binding loops of the pro tein.