Abstract
Annexins comprise a family of proteins that is possibly relevant to in io functions such as the formation of Ca2+ channels. The current work was undertaken to study the effect of annexin V (AxV) on vesicle fusion and/or destabilization, and on the phase behavior of the lipid in neutral and acidic conditions. Several techniques capable of providing information from lipid bilayer and multilayer systems were utilized to study the interaction of AxV with phosphatidylserine (PS) membranes as a function of Ca2+ concentration and pH. Microelectrophoresis indicates nearly complete binding of AxV to PS vesicles in the presence of Ca2+. Quenching of tryptophan fluorescence of AxV by doxyl radicals attached at different positions of the lipid chains indicates partial penetration of AxV near the glycerol region of the lipid. Dansyl fluorescence shows that the apparent relative permittivity in the headgroup region of the lipid decreases upon Ca2+ and pH-mediated binding of AxV. The additives AxV and Ca2+ cause the destruction of vesicles in the first place and their fusion only to a weaker degree at neutral pH. Phase behavior and details of headgroup structure were studied by IR spectroscopy. In neutral conditions ternary complex formation between the anionic binding sites of AxV, negatively charged PS headgroups and calcium ions stabilizes the gel state of the lipid. The effect of the divalent cations on the membranes is amplified in the presence of the macromolecules. At pH 5 AxV shifts the phase transition temperature of PS downwards. AxV destroys the integrity of PS vesicles in acidic conditions, even in the absence of Ca2+. These effects can be explained by an increased hydrophobicity of the protein at low pH. Binding of AxV to lipids becomes progressively driven by the hydrophobic effect with decreasing pH whereas the role of Ca2+-mediated interactions decreases. We suggest that a peripheral calcium mediated insertion mode of AxV at neutral pH shifts towards a more integral one at acidic pH.
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