The consequences of the binding of annexin V on the structure and dynamics of PC/PS bilayers were studied by means of fluorescence polarization, P-31 NMR, H-2 NMR, and fluorescence recovery after photobleaching (FRAP). Even at complete coverage of the lipid bilayers by the protein, annexin V showed no influence on the lipid molecular packing and the acyl chain flexibility of both PC and PS. The fluorescence polarization of the probe DPH, the P-31 NMR spectra, and deuterium quadrupolar splittings of P(d(31))OPS remained unchanged. However, upon binding of annexin V, two distinct populations of PC were visible in H-2 NMR, which were in slow exchange on the deuterium NMR time scale (microseconds), One component in the spectrum was identical to the protein-free sample, while a second, broad, component appeared. The presence of the protein induced a decrease in the transverse relaxation times (T-2e), indicative of the appearance of slow motions (milliseconds to microseconds), in the P(d(31))OPS spectrum and in the P(d(31))OPC broad component. FRAP experiments were carried out with the probes C-12-NBD-PC and C-12-NBD-PS: at saturation, annexin V reduced the lateral diffusion rate of PC by 40% and nearly blocked the diffusion of PS. These combined experiments are consistent with a model in which annexin V enters a proteolipidic complex in the form of an extended 2D network, stabilized by specific interactions with PS. As seen from the lateral diffusion rates and the acyl chains NMR spectral parameters, two separate lipid populations appear, presumably corresponding to those interacting with annexin V (PC and PS) and protein free domains (mainly PC).
