NMR in the study of ligand conformation and orientation within lipid membrane bilayers

Date of Completion

January 2004


Chemistry, Pharmaceutical|Health Sciences, Pharmacy|Biophysics, General




To explore drug-membrane interactions by high-resolution NMR, we have established a novel membrane bilayer model, the phospholipid bicelle system, and have investigated the conformational properties as well as the preferred orientations of two lipophilic cannabinoids (Δ8-THC and its O-methyl ether analog Me-Δ8-THC). While there are no observable conformational differences either in chloroform or in SDS micelle solutions, Δ8-THC tends to bend its pentyl tail towards the tricyclic ring system whereas Me-Δ8-THC's counterpart is extended in the bicelle membrane system. In addition, Δ8-THC orients itself in the membrane with its long axis perpendicular to the bilayer normal but Me-Δ8-THC orients with its long axis generally parallel to the bilayer normal. By using a magnetically oriented phospholipid bicelle preparation, we have also investigated the preferred orientation of 17β-estradiol with respect to the membrane bilayers. Our results highlight the advantages of using a bicelle preparation as a model membrane bilayer system in conducting ligand conformational analysis without the need for isotopic labeling. ^ Because of its high structural symmetry, the best 1H high-resolution spectrum from anandamide in a bicelle preparation is still severely overlapped, making conformational analysis impossible. We have then performed 13C-observed (31p or 2H-dephased) REDOR experiments with specifically 13C-labeled anandamide and 2H-labeled lipid molecules. The anandamide conformation has been studied by computing the internuclear distances from the REDOR data. Given that multiple intermolecular spin interactions are involved in our anandamide/lipid bilayer system, we have proposed a model to represent their geometric relationship and have written an efficient Fortran program to calculate the internuclear distances between the spin labels. Our results demonstrate that anandamide only adopts an extended conformation in the membrane although it may have many conformations in solution. ^ With the successful development of the bicelle membrane model for high resolution NMR and the application of solid-state REDOR NMR in deducing specific internuclear distances, our ability to determine the conformational properties and the preferred orientations of lipophilic ligands in membranes has been greatly advanced. These approaches should also be of more general value in studying the interactions of other small molecules, peptides, and integral membrane proteins with membranes. ^