Direct interaction between amphotericin B and ergosterol in lipid bilayers as revealed by 2H NMR spectroscopy.

Although amphotericin B (AmB) is thought to exert its antifungal activity by forming transmembrane ion-permeable self-assemblies together with ergosterol, no previous study has directly proven AmB-ergosterol interaction. To establish the interaction, we measured (2)H NMR using deuterium-labeled sterols and AmB. The (2)H NMR spectra of deuterated ergosterol in palmitoyloleoylphosphatidylcholine (POPC) bilayers showed that fast axial diffusion of erogosterol was almost completely inhibited by the coexistence of AmB. Conversely, cholesterol mobility in POPC membrane was essentially unchanged with or without AmB. These results unequivocally demonstrate that ergosterol has significant interaction with AmB in POPC bilayers. In addition, we examined the mobility of AmB using deuterium-labeled AmB, and found that, although AmB is almost immobilized in sterol-free and cholesterol-containing POPC membranes, a certain ratio of AmB molecules acquires mobility in the presence of ergosterol. The similar mobility of AmB and ergosterol in POPC bilayers confirmed the idea of the direct intermolecular interaction between ergosterol and AmB.

Conformation and location of membrane-bound salinomycin-sodium complex deduced from NMR in isotropic bicelles.

An ionophore antibiotic salinomycin was studied in a membrane environment consisting of isotropic bicelles, a better model for biological membranes than micelles, and its conformation and topological orientation in bicelles was determined. 2D NMR measurements and restrained conformational search revealed that salinomycin-sodium salt in bicelles adopts an open conformation in which the orientation of the E-ring is significantly different from that in crystal and solution structures. This conformational alteration breaks an intramolecular hydrogen bond between 28-OH and 1-O, dislocates the ether oxygen of the E-ring from a coordinated position to the sodium ion observed in the crystal, and consequently weakens the complexation between salinomycin and the sodium ion. Paramagnetic relaxation experiments using doxyl-phospholipids reveal that salinomycin is embedded shallowly in bicelles, with both terminals being closer to the water interface and the olefin portion facing the bicelle interior. Measurements of intermolecular NOEs between salinomycin and phospholipids further supported this orientation. Weaker complexation with sodium ion and positional preference in the membrane polar region may facilitate the catch-and-release of metal ions at the polar/nonpolar interface of bilayers. On the basis of these findings, a model for salinomycin-assisted transport of metal ions across biological membranes is proposed.

Detailed description of the conformation and location of membrane-bound erythromycin a using isotropic bicelles.

Although many nonpeptidic drugs target biological membrane and membrane proteins, it is still difficult to define the membrane-bound structure of the drugs. In this study, we utilized bicelles as a membrane model, since the bicelles, which have planar lipid bilayer portions, are thought to be a more appropriate and practical membrane model than micelles. Bicelles with a small diameter allow for measurements of liquid NMR due to fast tumbling in solution. We targeted erythromycin A (EA) as a membrane-binding compound because it is pointed out that the drug interacts with lysosomal membranes, inhibits phospholipase A, and consequently induces phospholipidosis as a side effect. The conformation of EA in the bicelle was successfully determined on the basis of coupling constants and NOEs. Measurements of intermolecular NOEs and paramagnetic relaxation times revealed that the drug is located shallowly in the membrane surface, with the dimethylamino group being close to the phosphate, and the macrolide portion adjacent to upper sides of the acyl chains. This study shows the general utility of isotropic bicelles for detailed conformational and orientational studies of membrane-associated nonpeptidic drugs.