Bilayer structure of ganglioside/cholesterol mixed system in the presence of Ca²⁺

We employed small-angle X-ray scattering measurement using synchrotron radiation to study structural properties of monosialoganglioside (G_M1)/cholesterol (chol) mixtures in aqueous dispersions, in the presence of 0-50 mM CaCl₂. Various molar ratios of G_M1/chol were examined from 1:0 to 1:5. Without CaCl₂ the radius of gyration (R_g) of the aggregate increased linearly with increasing cholesterol content up to G_M1/chol = 1/1. At cholesterol content above G_M1/chol = 1/1, R_g of the aggregate was almost constant, and a sharp diffraction peak at q = 0.18 Å^-1, characteristic of cholesterol monohydrate crystals, was observed, suggesting the solubility limit of cholesterol in G_M1 aggregates is about G_M1/chol = 1/1. For the samples containing cholesterol of G_M1/chol = 1/0.6 and above, the diffraction patterns showed a set of peaks that correspond to ~85 Å periodic lamellar structures in the presence of CaCl₂ at concentrations higher than ~10 mM. The observed lamellar peak positions and the intensity ratio of the lamellar peaks (1st-4th) was not changed by the addition of CaCl₂ up to 100 mM, demonstrating once formed multilayered structure of G_M1/chol was not altered under the present conditions. Electron density profiles for this G_M1/chol bilayer were obtained. The Ca²⁺-induced vesicle-to-lamellar phase separation of this system was found to be highly reversible by the Ca²⁺ chelation with ethylenediamine-tetraacetic acid.