The Physical State of Hydrocarbons and Chlorophyll a Incorporated in Phospholipid Bilayers as Determined by Nuclear Magnetic Resonance Spectroscopy

Several classes of non-protein lipophilic molecules interact with biological membranes in physiologically important ways. Nuclear magnetic resonance has been used to investigate the physical state of two model membrane systems composed of a phosphatidylcholine and a lipophile. The motional state of straight-chain hydrocarbons in phospholipid bilayers has been studied by deuterium NMR. Wide-line 2H spectra have been obtained for multilamellar dispersions of dipalmitoylphosphatidylcholine containing perdeuterated hydrocarbons and fatty acids ten and sixteen carbons in length. Hydrocarbon spectra show three or four resolved quadrupolar splittings as well as a central maximum whose intensity increases with hydrocarbon concentration. The maximum quadrupolar splitting is two to three times less than that observed for fatty acids. These data indicate two pools of hydrocarbon in the bilayer which are in slow exchange on the 2H NMR timescale. The central maximum corresponds to hydrocarbons in an isotropic motional state and may be assigned to micro-droplets of hydrocarbon between the two monolayers of the bilayer. The various quadrupolar splittings indicate a flexibility gradient along the hydrocarbon chain and may be assigned to hydrocarbon lying parallel to the fatty acid chains of the lipid. The magnitude of the splittings, and the fact that the two ends of the hydrocarbon show equivalent splittings may be accounted for by either a rapid end-for-end flip of the molecule within one monolayer or sliding of the molecule between monolayers of the bilayer. To conduct these experiments, a 2H NMR spectrometer for observation of wide-line spectra utilizing a solid-echo pulse sequence and quadrature phase detection has been constructed. Chlorophyll-containing phospholipid bilayers have been investigated as a model system for light-harvesting antenna chlorophyll in vivo. The red shift of the chlorophyll visible absorption band observed in vivo is best duplicated by the model system at temperatures below the gel to liquid crystalline phase transition of the bilayer. The phase diagram of the two component bilayer as determined by differential thermal analysis shows a double eutectic with compound formation around 40 mole% chlorophyll. 31P, 13C and 1H magnetic resonance measurements confirm that phase separation occurs below the transition temperature and suggest that in the compound phase the chlorophyll Mg is coordinated by the phosphate of the lipid headgroup. The constant composition of the compound phase, the interaction of chlorophyll with lipid and the possibility of spatial ordering of chlorophylls in the compound make it an attractive antenna model.