Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior

Abstract

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.

Solid‐state 2H NMR studies of the effects of cholesterol on the acyl chain dynamics of magnetically aligned phospholipid bilayers

“…9 The rate of rotation and the internal motions of cholesterol have also been determined by 13 C NMR measurements, revealing that the hydrophobic tail of cholesterol undergoes rapid rotational motional between different conformations. 10 The orientation of cholesterol in phospholipid bilayers, with its polar OH group encountering the aqueous phase and the hydrophobic steroid ring oriented parallel to and buried in the hydrocarbon chains of phospholipids, has previously been confirmed by x-ray diffraction and neutron diffraction studies. 11 The data from such studies indicate that cholesterol is located such that the polar hydroxyl group is in the immediate vicinity of the headgroup of the phospholipid acyl chain.…”

“…This property is often referred to as the condensing effect of cholesterol, which occurs at a temperature above the gel-to-liquid crystalline phase transition of the phospholipids and a disordering effect below the gel-to-liquid crystalline phase transition. 10,13,14 In a pure phospholipid membrane, the average orientation of the hydrocarbon chains defines the structure of the membrane, whereas the fluctuations in the polar headgroups impose different segmental motions that define the reorientational dynamics of the bilayer. 15 These properties can be addressed if the segmental order parameters and the T 1 and T 2 relaxation times are measured using deuterated phospholipids.…”

Sterols and membrane dynamics

“…3a). The motional processes responsible for maintaining such a state (molecular rotation, trans-gauche bond isomerization) occur in the microsecond-nanosecond time scale and gently increase in speed with increasing temperature [15]. The lipid bilayer is thus maintained in a liquid-ordered state (lo), where molecules still undergo rotational and lateral diffusion in the membrane but to a lower extent.…”

Macroscopic Orientation of Natural and Model Membranes for Structural Studies

“…The complete secondary structure of gram icidin and its dynamic properties in membranes have also been obtained using 2H and 15N NMR (6-9). In addition, the complete structure and orientation of deu terated retinal in bacteriorhodopsin at different states of its photocycle has been resolved (10), The average molecular and orientational order of lipids and peptides in liquid crystalline membranes could also be deter mined unambiguously in this way (11)(12)(13)(14).…”