Broadline 1H and 2H nuclear Magnetic resonance studies of binary lipid mixtures containing docosahexaenoic acid
Creators: name: Lloyd-Smith, James
Publisher: name: University of British Columbia
Year: 2009

description: Containing six double bonds, docosahexaenoic acid (DHA) is the most highly unsaturated acyl chain commonly found in biological membranes. It is present in large proportions in retinal and synaptic membranes and there is substantial evidence that specific DHA concentrations are required for proper function of some membrane proteins. A prevalent hypothesis attributes this functional influence to the supposed wedge shape of polyunsaturated lipid molecules, suggesting that lipids with DHA chains influence conformational changes of membrane proteins by exerting pressure laterally through the centre of the bilayer. This model was tested using broadline ¹H- and ²H-NMR techniques to study a series of binary lipid mixtures. The membrane systems were mixtures of monounsaturated l-palmitoyl-d₃₁-2-oleoyl-phosphatidylcholine (POPC-d₃₁) and polyunsaturated l-palmitoyl-d₀-2-docosahexaenoyl-phosphatidylcholine (PDPC-d₀) in varying proportions (from Omol% PDPC to 100mol% PDPC), and were designed to be representative of membranes found in the brain. The deuterated POPC acted as a reporter molecule in ²H-NMR experiments, giving a clear picture of the effects of increasing DHA content on the surrounding lipid matrix. The ¹H-NMR signal included contributions from both lipid species, and yielded information regarding general properties of the membrane. Spectroscopic measurements were made and analyzed in terms of orientational order parameters of the lipid acyl chains, and relaxation experiments yielded further information on membrane dynamics at a range of timescales. All experiments were conducted at 10° C, 25° C, and 37° C. The order parameter measurements showed remarkably little change across the full range of membrane compositions. As PDPC content increased, the residual second moment (which is proportional to a mean-squared orientational order parameter) of the proton spectrum increased slightly, while that of the deuterium line showed a small decrease. We infer that the average chain length and order of PDPC molecules is similar to that of POPC-d₃₁, and possibly that species-enriched microdomains may have formed which isolated POPC from the effects of increasing polyunsaturation. The ²H-NMR order parameter profiles demonstrated clearly that the addition of PDPC influenced the conformational freedom of POPC-d₃₁'s perdeuterated chain. A slight decrease in orientational order was observed in the lower half of this chain, indicating greater mobility in that region. This is the first direct measurement of the effect of DHA-bearing lipids on the the chain order of other, less unsaturated lipids in.the membrane, and is not consistent with the prediction of the "wedge model" that lateral pressure increases in the centre of the bilayer. To explain this observation, it is suggested that the DHA chains spend slightly more time doubled back towards the lipid-water interface, vacating space deep in the bilayer which is occupied by other acyl chains. T₁[sub z] values for both ¹H- and ²H-NMR were found to rise significantly as PDPC was added to the membrane. Since T₁[sub z] also increased with temperature, this is deduced to result from a decrease in the correlation times of very fast motions. A likely example, which is consistent with the above model for DHA's motions, would be speeding up of the trans-gauche isomerizations of the acyl chains. Finally, T₂[sup qe] values for POPC-d₃₁'s perdeuterated chain were found to decrease markedly as PDPC was added, dropping by almost a factor of two between the 0% and 75% PDPC samples. T₂[sup qe] relaxation processes are determined to arise from thermally-activated collective motions of the bilayer, and the primary mechanism is proposed to be membrane surface undulations. If the twofold change in T₂[sup qe] resulted entirely from increased amplitude of these fluctuations, then the membrane curvature energy KC has been reduced by almost 50% by the addition of 75 mol% PDPC to the POPC – d₃₁ membrane; the real effect is expected to be somewhat smaller, since other processes doubtless contributed to the observed change. The relation of the order parameter measurements to the proposed decrease in curvature energy is considered, and the implications of these results for DHA ' s role in membrane function are discussed. While significant effects can be postulated for increased flexibility or altered chain packing, no explanation has emerged for the extreme specificity with which nature chooses DHA for certain important systems.