Clustering of Stearic Acids in Model Phospholipid Membranes Revealed by Double Electron–Electron Resonance

Free fatty acids play various important roles in biological membranes. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) of spin-labeled biomolecules is capable of studying magnetic dipole-dipole (d-d) interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to study intermolecular d-d interactions between doxyl-spin-labeled stearic acids (DSA) in gel-phase phospholipid bilayers composed either of an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine or of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. DEER data obtained for different DSA concentrations showed that DSA molecules at their concentration in the bilayer χ larger than 0.5 mol % are assembled into lateral lipid-mediated clusters, with a characteristic intermolecular distance of 2 nm. Some evidences were obtained indicating that clusters may consist of "subclusters", alternatively appearing in two opposite leaflets. Conventional electron paramagnetic resonance (EPR) spectra for the gel-phase bilayers showed that for χ larger than 2 mol % the molecules in the clusters stick together, forming oligomers. Room-temperature EPR spectra for the liquid-crystalline phase were found to change noticeably for χ larger than 0.5 mol %, which may indicate the clustering in a liquid-crystalline phase similar to that observed by DEER in the gel phase.