THE CALCIUM-DEPENDENT BINDING OF ANNEXIN-V TO PHOSPHOLIPID-VESICLES INFLUENCES THE BILAYER INNER FLUIDITY GRADIENT

The fluidity of the hydrophobic interior of phospholipid vesicles afte r calcium-dependent binding of human annexin V (AVH) was studied using EPR spectroscopy. Vesicles (SUVs) composed of PC or PE and an acidic phospholipid (alternatively PS, Pa, or CL) were probed at different bi layer depths by either phosphatidylcholine, or the accompanying acidic phospholipid, bearing a spin label probe at position C-5, C-12, or C- 16 of the sn-2 acyl chain. Alternatively, the vesicle surface was prob ed with a polar head spin labeled PE (PESL). The EPR spectra of annexi n-bound bilayer domain(s) were obtained by computer spectral subtracti on. The order parameter values (S) from the resulting difference spect ra revealed that the bilayer hydrophobic interior has a greatly altere d fluidity gradient, with an increased rigidity up to the C-12 positio n. Thereafter, the rigidification progressively vanished, The effect i s not linked to the phospholipid class, since all the acidic phospholi pid spectra, as well as phosphatidylcholine, shared the same sensitivi ty to the bound protein. The observed membrane rigidification appears to parallel the ''crystallizing'' tendency of vesicle-bound annexin V, but may not be involved in the calcium channeling activity of this pr otein.