Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane

Annexin V is of crucial importance for detection of the phosphatidylserine of apoptotic cell membranes. However, the manner in which different amounts of phosphatidylserine at the membrane surface at different stages of apoptosis contribute to binding of annexin V is unclear. We have used a quartz crystal microbalance combined with dissipative monitoring (QCM-D) and neutron reflectivity to characterize binding of human annexin V to supported bilayers of different phospholipid composition. We created model apoptotic bilayers of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine (POPS) in the ratios 19:1, 9:1, 6.7:1, 4:1, 3:1, and 2:1 (w/w) in the presence of 2.5 mM CaCl2. QCM-D data revealed that annexin V bound less to supported fluid lipid bilayers with higher POPS content (>25 POPS). Neutron reflectivity was used to further characterize the detailed composition of lipid bilayers with membrane-bound annexin V. Analysis confirmed less annexin V binding with higher POPS content, that bound annexin V formed a discrete layer above the lipid bilayer with little effect on the overall structure of the membrane, and that the thickness and volume fraction of the annexin V layer varied with POPS content. From these results we show that the POPS content of the outer surface of lipid bilayers affects the structure of membrane-bound annexin V.