Phospholipid head group dynamics have been studied by pulsed phosphorus-31 nuclear magnetic resonance (31P-NMR) of unoriented and macroscopically aligned dimyristoylphosphatidylcholine model membranes in the temperature range, 203-343 K. Lineshapes and echo intensities have been recorded as a function of interpulse delay times, temperature and macroscopic orientation of the bilayer normal with respect to the magnetic field. The dipolar proton-phosphorus (1H-31P) contribution to the transverse relaxation time, T2E, and to lineshapes was eliminated by means of a proton spin-lock sequence. In case of longitudinal spin relaxation, T1Z, the amount of dipolar coupling was evaluated by measuring the maximum nuclear Overhauser enhancement. Hence, the results could be analyzed by considering chemical shift anisotropy as the only relaxation mechanism. The presence of various minima both in T1Z and T2E temperature plots as well as the angular dependence of these relaxation times allowed description of the dynamics of the phosphate head group in the 31P-NMR time window, by three different motional classes, i.e., intramolecular, intermolecular and collective motions. The intramolecular motions consist of two hindered rotations and one free rotation around the bonds linking the phosphate head group to the glycerol backbone. These motions are the fastest in the hierarchy of time with correlation times varying from less than 10(-12) to 10(-6) s in the temperature range investigated. The intermolecular motions are assigned to phospholipid long axis rotation and fluctuation. They have correlation times ranging from 10(-11) s at high temperatures to 10(-3) s at low temperatures. The slowest motion affecting the 31P-NMR observables is assigned to viscoelastic modes, i.e., so called order director fluctuations and is only detected at high temperatures, above the main transition in pulse frequency dependent T2ECP experiments. Comprehensive analysis of the phosphate head group dynamics is achieved by a dynamic NMR model based on the stochastic Liouville equation. In addition to correlation times, this analysis provides activation energies and order parameters for the various motions, and a value for the bilayer elastic constant.

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Multipulse dynamic NMR has been employed to study molecular order and dynamics of deuteron (2H) labeled phospholipid membranes. Variation of pulse sequence and pulse separation provides the large number of independent experiments necessary for a proper molecular characterization of the systems. Analysis of these experiments is achieved by employing a density matrix formalism, based on the stochastic Liouville equation. Arbitrary relaxation rates and line shapes of single and multiple quantum transitions are considered. The various 2H NMR experiments of macroscopically unoriented bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), specifically deuterated at the 6- and 14-position of the 2-chain, are faithfully reproduced by the model. Computer simulations provide the orientational distributions and conformations of the hydrocarbon chains and the correlation times of the various motions. In the Lα phase the correlation times τR∥ and τR⊥ for chain rotation and chain fluctuation are of the order of 10−8 s, while trans–gauche isomerization occurs significantly faster (τJ∼10−10 s). At the main transition all chain motions slow down abruptly. Further cooling in the Pβ′ phase first continuously decreases the motions. However, 10 K below the pretransition (hysteresis), there is another abrupt slow down of the chain dynamics. In the Lβ′ phase at T=265 K all three motions occur with correlation times of 10−6 to 10−5 s. Because of higher activation energies, however, intermolecular chain motions freeze out first on the time scale of a particular NMR experiment. Thus, at temperatures T<210 K, trans–gauche isomerization becomes the dominant process. Detection of this motion is possible even at T=168 K, where τJ is of the order of 10−4 s. Arrhenius plots of the various correlation times provide the motional activation energies. Values of 9<EJ<14 kJ/mol for trans–gauche isomerization correspond to the local character of this process. As expected, the activation energies for chain rotation (50<ER∥ <69 kJ/mol) and chain fluctuation (53<ER⊥ <79 kJ/mol) are substantially higher. The correlation times for methyl group rotation form a continuous straight line on the Arrhenius plot throughout the three phases studied, yielding an activation energy of EJ(CD3) =9.9 kJ/mol. Molecular order of the chains is discussed in terms of two parameters SZZ and SZ′Z′, characterizing the orientational order of the chains as a whole and the conformational order at a particular segment. In the Lα phase the hydrocarbon chains are partially disordered (0.44<SZZ <0.6) and melted, exhibiting segmental order parameters of SZ′Z′ (C-6)∼0.75 and SZ′Z′ (C-13)∼0.35, respectively. As expected, conformational order decreases from the central unit to the terminal one (order gradient). The Pβ′ phase exhibits two different chain order parameters of SZZ ∼0.6 and SZZ ∼0.9, indicating heterogeneous chain packing. A unique structural interpretation of this result is not yet possible since the microscopic heterogeneity is compatible with most proposed models. In the Lβ′ phase we find SZZ >0.95, SZ′Z′ (C-6)>0.95, and SZ′Z′ (C-13)>0.9, consistent with highly ordered, fully extended hydrocarbon chains.

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The anisotropy and pulse frequency dispersion of the spin–spin relaxation time TCP2E from Carr–Purcell–Meiboom–Gill pulse sequences is employed to evaluate the major contribution to transverse 2H spin relaxation in bilayer membranes. Analysis of the experiments is achieved in terms of a density operator formalism, employing the stochastic Liouville approach. From a comparison of the observed angular and frequency dependences of TCP2E with those predicted for order director fluctuations, we conclude that collective lipid motions constitute the dominant transverse relaxation process. Computer simulations provide the viscoelastic parameters of the lipid membranes. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers at T=318 K an average elastic constant of K=2×10−11 N and an effective viscosity of η=0.1 P have been determined. Using the experimentally accessible value for the long wavelength cutoff of the elastic modes, one obtains the mean square amplitude of the director fluctuations 〈θ20〉=0.04. This corresponds to an order parameter of SOF=0.94. Apparently, the contributions of the collective motions to the measured order parameters are marginal.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

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