Organization and dynamics of the proteolipid complexes formed by annexin V and lipids in planar supported lipid bilayers.
The consequences of the binding of annexin V on its lateral mobility and that of lipids were investigated by means of experimental and simulated FRAP experiments. Experiments were carried out on planar supported bilayers (PC/PS 9:1 mol/mol mixtures) in the presence of 1 mM CaCl2 in the subphase. The probes C12-NBD-PS and fluorescein-labeled annexin V were used and the data compared with that previously obtained for C12-NBD-PC [Saurel, O., Cézanne, L., Milon, A., Tocanne, J. F., & Demange, P. (1998) Biochemistry 37, 1403-1410]. At complete coverage of the lipid bilayer by the protein (Cannexin = 80 nM), the lateral mobility of C12-NBD-PC was reduced by 40% while C12-NBD-PS and bound annexin V molecules were nearly immobilized (D < 10(-)11 cm2/s). At moderate protein concentration (20 nM < Cannexin < 80 nM), best fitting of the lipid and protein probe recoveries was achieved with one single diffusion coefficient and a mobile fraction close to 100%, indicating homogeneous lipid and protein populations. In contrast, at low protein concentration (Cannexin < 20 nM), C12-NBD-PS showed a two-component diffusion. The slow PS population at Cannexin < 20 nM and the single PS population at Cannexin > 20 nM moved at the same rate that bound annexin V (mobile fraction close to 100%), indicating strong PS/protein interactions. With the aid of computer simulations of the lateral motion of PC molecules, based 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 rather simple model of a proteolipidic complex consisting of an extended 2-D crystalline protein network facing the lipid bilayer and stabilized by strong interactions between annexin V and PS molecules. In this model, immobilization of annexin V and PS molecules originates from their mutual interactions. The slowing down of PC molecules is due to various obstacles to their lateral 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 hydrophobic amino acid residues located on the AB calcium-binding loops of the protein.
Institut de Pharmacologie et de Biologie Structurale du CNRS, Toulouse, France.
L
L
Cézanne
A
A
Lopez
F
F
Loste
G
G
Parnaud
O
O
Saurel
P
P
Demange
J F
JF
Tocanne
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry*; D002417:Cattle; D003198:Computer Simulation; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010718:Phosphatidylserines / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry*; D013816:Thermodynamics
D017304:Annexin A5; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D010718:Phosphatidylserines; D011510:Proteolipids; C068204:1-palmitoyl-2-(N-(12-((7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino)dodecanoyl))phosphatidylserine