Deuterium Nuclear Magnetic Resonance Spectroscopic Study of the Fluorescent Probe Diphenylhexatriene in Model Membrane Systems

We have investigated the deuterium (²H) nuclear magnetic resonance (NMR) spectra of two ²H-labeled fluorescence probes (trans,trans,trans-1,6-diphenylhexa-1,3,5-trienes, DPHs) incorporated into model lipid bilayer membrane systems at various temperatures. The membranes consisted of multilamellar bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing varying concentrations of cholesterol. The conventional one-order parameter approach often used in the analysis of the NMR data of lipid membranes does not explain the observed temperature variations of the spectral features. Consistent with the molecular symmetry, the results have thus been analyzed in terms of an ordering matrix with more than one independent element. The molecular order parameter (S_NMR), the order along the long molecular axis, in the pure lipid system varies from 0.49 to 0.26 as the temperature is increased from 25 to 57 °C. These values are somewhat larger than the order parameters obtained from fluorescence depolarization (S_FLU) on sonicated DMPC vesicles. Such discrepancies probably arise from the looser packing of the sonicated vesicles. Addition of cholesterol to the model membranes causes the order parameter of the probe molecules to increase. At 35 °C, S_NMR increases from 0.38 (with no cholesterol) to 0.92 (in the presence of 50 mol % cholesterol). These values are about 10% larger than those obtained from fluorescence depolarization studies on sonicated vesicles. The S_NMR for DPH are somewhat larger than those obtained in earlier NMR studies of ²H-labeled cholesterol. However, they compare well with those obtained for ²H-labeled DMPC. These results suggest that ²H-labeled DPH, and by analogy DPH itself, can be a good probe for following the molecular ordering of lipids and for studying lipid-sterol interactions. We believe these studies should lay the groundwork for future studies of DPH/lipid chain organization in systems containing polypeptides and proteins.