Anisotropic motion and molecular dynamics of cholesterol,lanosterol, and ergosterol in lecithin bilayers studied by quasi-elastic neutron scattering

Quasi-elastic neutron scattering (QENS) was employed to study the molecular dynamics of three structurally related sterols, namely, cholesterol, lanosterol, and ergosterol. Oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) were investigated at 40 mol % sterol content and at three temperatures (20, 36, and 50 degrees C) for two energy resolutions. Data analysis was concentrated on a direct comparison of the out-of-plane and the in-plane high-frequency motions of the three sterols in terms of their rates and amplitudes. The (spatially restricted) diffusive motion of the three sterols in the two directions was characterized by diffusion constants in the range of (5-30) x 10(-12) x m(2) x s(-1), with a significantly faster rate of diffusion along the membrane normal, resulting in a diffusional anisotropy, D(a). At low temperature (20 degrees C), cholesterol showed the highest value (D(a) = 4.5), while lanosterol gave the lowest one (D(a) = 2.0). At high temperature (50 degrees C), ergosterol diffusion had the highest diffusion anisotropy (D(a) = 2.0) compared to lanosterol (D(a) = 1.8) and cholesterol (D(a) = 1.6). Most interestingly, cholesterol showed at all three temperatures an amplitude of its out-of-plane-motion of 1.0-1.1 nm, more than a factor of 3 higher than measured for the other two sterols. This finding suggests that the short alkyl chain of the cholesterol molecule may cross at high frequency the bilayer midplane, while the other two sterols remain confined within the geometrical limits of each monolayer leaflet. The results provide an example of how slight structural alterations of sterols can affect their molecular dynamics in bilayers, which in turn may be relevant to the membrane micromechanical properties.     

Molecular motion of small nonelectrolyte molecules in lecithin bilayers

Summary We have used magnetic resonance spectroscopy, both ESR and¹³C spin relaxation, to measure translational and rotational mobilities and partition coefficients of small nitroxide solutes in dipalmitoyl lecithin liposomes. Above the bilayer transition temperature,T _c, the bilayer interior is quite fluid, as determined from the solutes' rapid rotational and moderately rapid translational motion; the rotational and translational viscosities within the bilayer are _R <1cP and =6–10cP, respectively. and _R are independent of molecular size for all solutes studied, but all were small compared to the size of the phospholipids. , and probably _R , are relatively independent of temperature aboveT _c, but both increase very sharply as temperature is lowered belowT _c; at 32°C, _R increases to 6cP and is greater than 1000 cP. Anisotropy of rotational motion increases gradually as temperature is lowered toT _c, and changes little belowT _c; anisotropy of translational motion was not investigated.¹³C nuclear spin relaxation measurements indicate that translational motion of nitroxide solutes is more rapid in the center of the bilayer than near the polar interface. It takes at least 100 nsec for a solute molecule to cross the bilayer/water interface. We estimate a lower limit of 2 sec/cm for the interfacial resistance to solute diffusion; this result indicates that interfacial resistance dominates permeation across the membrane. The relative solubility, or partition coefficient, is a strong function of solute structure, and decreases abruptly on cooling through the transition temperature. From the partition coefficient and its temperature dependence we calculate the free energy, enthalpy, and entropy of partition. Effects of cholesterol on partition and diffusion coefficients are compatible with the interpretation that bilayers containing cholesterol consist of two phases.     

Glycerolipids: common features of molecular motion in bilayers

In the present study, analysis of 2H NMR line-shape and spin-lattice relaxation behavior has been used to investigate the dynamics of several glycolipid and phospholipid bilayers. The gel-phase spectra of these lipids labeled at the C3 position of the glycerol backbone are broad (approximately 90 kHz) and characteristic of fast-limit axially asymmetric motion. Moreover, anisotropic spin-lattice relaxation is observed in all of these systems. The line-shape and relaxation features of the lipids in the gel phase were best simulated by using a fast-limit three-site jump model, with relative site populations of 0.46, 0.34, and 0.20. This motion is associated with an internal jump about the C2-C3 bond of the glycerol backbone. A second motion, rotation about the long axis of the molecule, is needed to account for the observed temperature dependence of the quadrupolar echo amplitude and the spectral line shape above and below the gel to liquid-crystalline phase transition temperature. On the other hand, the gel-phase spectra of phospholipids labeled at the C2 position of the glycerol backbone are also characterized by a fast internal motion, which is simulated by a two-site librational jump. The results indicate that the glycerol backbone dynamics of the glycolipid and phospholipid systems investigated in this study can be described in terms of common fast internal motions and a slower whole molecule axial motion. These results are compared with previous dynamic studies of similar systems.     

Solvation of oxygen in lecithin bilayers.

The solubility of oxygen in dipalmitoyllecithin (DPL) and paraffinc C19 has been investigated by measurement of the enhanced proton relaxation rates under the influence of oxygen pressure. The paraffin shows a noticeable effect in the rotator phase, but not so in the crystalline phase. tin contrast to paraffins, both phases of DPL-bilayers dissolve oxygen, but the solubility in the liquid-crystalline phase is greater than in the crystalline state by a factor approximately equal to 3. Furthermore, the experiments indicate a distribution of electron relaxation times in the crystalline phase in contrast to the liquid-crystalline phase. A possible explanation of this behaviour is a multiphase structure of the "crystalline' lamellae. The biological relevance of thbse results could be a triggering of the gas-transport by the alveolar lining of lungs, if cyclic phase transitions occur during the breathing-cycle.     

The molecular reorganization of lipid bilayers by osmium tetroxide. A spin-label study of orientation and restricted y-axis anisotropic motion in model membrane systems

Phospholipid dispersions spontaneously form oriented lamellar multilayers when dried onto glass slides. These oriented multilayers form useful model systems for studying the molecular dynamics of lipid bilayers. In order to examine the effects of osmium tetroxide on the orientation and motion of hydrocarbon chains in lipid bilayers, lecithin multilayers containing the spin label 3-doxyl-5α-cholestane (the 4′,4′-dimethyloxazolidine-N-oxyl derivative of 5α-cholestan-3-one) were prepared and examined by electron spin resonance spectroscopy. In egg lecithin multilayers at room temperature and 81% relative humidity the osmium tetroxide causes nearly complete loss of orientation and severe reduction of molecular motion. In contrast, the high degree of order in l-α-dipalmitoyl lecithin multilayers is not affected by exposure to osmium tetroxide vapors. Experiments are also reported on macroscopically disordered lecithin preparations, and the data support the conclusions drawn from the ordered lecithin multilayers that rotational mobility of the probe is severely reduced by fixation of the lipid chains.A simple mathematical model has been developed to account for the amplitude of the high-frequency (τ < 10^?8 sec) restricted y-axis anisotropic motion occurring in the bilayer plane. Since the y-axis is roughly parallel to the molecular axis of the rigid steroid spin label, this model enables quantitative comparisons of various degrees of restricted motion about the molecular axis.     

Thermal phase transitions in deuterated lecithin bilayers.

Differential Thermal Analyses and Optical Density measurements show that perdeuteration of the fatty acid residues in phosphatidylcholines causes a 4–5°C decrease in the phase transition temperatures of bilayer dispersions prepared from these deuterated phospholipids. The implications of these findings on the use of deuterated phospholipids in membrane research will be briefly discussed.     

X-ray diffraction studies of lecithin bilayers.

Counting the individuals in a population before and after an annual period of environmental stress allows the proportion s of the initial population surviving the period to be computed. A series of such observations over n annual periods gives a sequence s₁, s₂, …, s_n. A statistical model is formulated from axioms describing the survival process, and it is concluded that these observed values may usefully be regarded as realizations of a random variable that arises from the normal generated distribution (n.g.d.). Equations for estimating the n.g.d. parameters ζ and τ² from observed survival proportions by the method of moments and maximum likelihood are given. The distributions of parameter estimates ζ and τ² are obtained and discussed in the context of testing hypotheses comparing survival among different populations. Finally, the dependence of the n.g.d. upon parameters ζ and τ² is examined in terms of altering survival, either by population self-regulation mechanisms or man-induced controls. The intent is to provide insight into the relationship between the n.g.d. and its supporting axioms and, more generally, basic knowledge of population processes.     

Ion and sugar permeabilities of lecithin bilayers: Comparison of curved and planar bilayers

Summary Na⁺ and sugar permeabilities of egg lecithin bilayers were measured using curved bilayers and planar bilayers as represented by single-bilayer vesicles and black lipid films, respectively. The Na⁺ permeability coefficient measured with single-bilayer vesicles at 25°C is (2.1±0.6)×10^–13 cm sec^–1. Because of technical difficulties it has been impossible to measure ionic permeabilities of values lower than about 10^–10 cm sec^–1 in planar (black) lipid bilayers using tracer methods. Thed-glucose andd-fructose permeabilities were measured with both curved and planar bilayers. The permeability coefficients measured with vesicles at 25°C are (0.3±0.2)×10^–10 cm sec^–1 for glucose and (4±1)×10^–10 cm sec^–1 ford-fructose; these are in reasonable agreement with the corresponding values obtained for planar (black) lipid bilayers which are (1.1±0.3)×10^–10 cm sec^–1 ford-glucose and (9.3±0.3)×10^–10 cm sec^–1 ford-fructose, respectively.This paper is dedicated to the memory of Walther Wilbrandt,cuius nomini nullum par elogium.     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers

Summary The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8-ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio /gt higher than 1,8-ANS ( being the rotational relaxation of in plane rotations and the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.Abbreviations 2,6-MANS 2-n-methylanilinonaphthalene-6-sulfonate - 1,8-ANS 1-anilinonaphthalene-8-sulfonate     

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers.

The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8 ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio rho/tau higher than 1.8-ANS (rho being the rotational relaxation of in plane rotations and tau the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.     

Anisotropic motion of cholesterol in oriented DPPC bilayers studied by quasielastic neutron scattering: the liquid-ordered phase.

Quasielastic neutron scattering (QENS) at two energy resolutions (1 and 14 microeV) was employed to study high-frequency cholesterol motion in the liquid ordered phase (lo-phase) of oriented multilayers of dipalmitoylphosphatidylcholine at three temperatures: T = 20 degrees C, T = 36 degrees C, and T = 50 degrees C. We studied two orientations of the bilayer stack with respect to the incident neutron beam. This and the two energy resolutions for each orientation allowed us to determine the cholesterol dynamics parallel to the normal of the membrane stack and in the plane of the membrane separately at two different time scales in the GHz range. We find a surprisingly high, model-independent motional anisotropy of cholesterol within the bilayer. The data analysis using explicit models of molecular motion suggests a superposition of two motions of cholesterol: an out-of-plane diffusion of the molecule parallel to the bilayer normal combined with a locally confined motion within the bilayer plane. The rather high amplitude of the out-of-plane diffusion observed at higher temperatures (T >/= 36 degrees C) strongly suggests that cholesterol can move between the opposite leaflets of the bilayer while it remains predominantly confined within its host monolayer at lower temperatures (T = 20 degrees C). The locally confined in-plane cholesterol motion is dominated by discrete, large-angle rotational jumps of the steroid body rather than a quasicontinous rotational diffusion by small angle jumps. We observe a significant increase of the rotational jump rate between T = 20 degrees C and T = 36 degrees C, whereas a further temperature increase to T = 50 degrees C leaves this rate essentially unchanged.     

Measurement of the curvature-elastic modulus of egg lecithin bilayers

The first determination of the curvature-elastic modulus k of a bilayer is presented. The method is based on the microscopic study of thermally fluctuating bilayer tubes. For egg lecithin at room temperature we obtain k = (2.3 ± 0.3) · 10⁻¹²erg.     

Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded β-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the β-barrel and loop regions were probed by the spin-lattice relaxation times of the C(α) and C(β) atoms of the serine and threonine residues, and by cross-polarization dynamics. The β-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.     

Diffusion within egg lecithin bilayers resembles that within soft polymers

An analysis is presented of how the permeability coefficient/octanol:water partition coefficient ratio for 33 different chemical substances crossing egg lecithin bilayers depends on the molecular volume of the substances. From this analysis we conclude that bilayers made from egg lecithin behave as soft polymers in their discrimination between permeants of different sizes and shapes.     

Phase diagrams of bilayers of dimyristoyl lecithin plus heteroacid lecithins

Aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) mixed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) or with 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC) have been studied by differential scanning calorimetry. Analysis of phase diagrams of the resulting bilayers indicated that both POPC-DMPC were miscible, but not ideally mixed, in the gel and in the liquid-crystalline states. In both states, estimated values for excess interaction energies were more positive for PSPC-DMPC than for POPC-DMPC, indicating that mixing was less ideal in the former system.