Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: effects of unsaturation and hydration

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852-860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe molecules do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.     

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.     

Radioprotective effects of lipid A, liposomes, and liposomes containing lipid A in mice

Lipid A from Gram-negative bacterial lipopolysaccharide (endotoxin) was incorporated into liposomal membranes and examined as a prophylactic radioprotectant compound in lethally irradiated mice. Splenic hematopoietic activity, resulting in increased numbers of spleen cell colonies, was induced both by lipid A alone or more strongly by liposomal lipid A. Increased survival of lethally irradiated animals was induced to a slight extent by liposomes alone, to a greater extent by lipid A, and at the highest level by liposomes containing lipid A. Under conditions where 100% of untreated or saline-treated animals died of acute radiation syndrome after 20 days, more than 90% of the animals pretreated with liposomal lipid A were still alive 30 days after irradiation. We conclude that lipid A had substantial radioprotectant activity by itself, and the activity was enhanced by incorporation into liposomes. Liposomes alone also exhibited mild radioprotectant effects.     

Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles

The preparation of a new kind of multilayered liposome, called a stable plurilamellar vesicle (SPLV), is described. Although SPLVs and classical multilamellar vesicles (MLVs) are made of the same materials and appear overtly similar in the electron microscope, the two types of vesicles differ as determined by stability, entrapment efficiency, electron spin resonance (ESR), NMR, X-ray diffraction, and biological effects. It is demonstrated that, contrary to what has been assumed, classical MLVs exclude solutes during their formation and, thus, are under a state of osmotic compression. By contrast, the SPLV process produces liposomes that are not compressed. The effects of osmotic compression are discussed. It is suggested that the state of osmotic stress is an important variable that distinguishes various types of liposomes and that has significant physical and biological consequences.     

Location and orientation of DODCI in lipid bilayer membranes: effects of lipid chain length and unsaturation.

The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12>C14>C16>C18C20) and in unsaturated lipids (C14:1>C14:0, C16:1>C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule in the interior region of the membrane is more when the structure of th bilayer is more fluid-like.     

Rotational dynamics of surface probes in lipid vesicles

Translational and rotational diffusion of fluorescent molecules on the surface of small biological systems such as vesicles, proteins and micelles depolarize the fluorescence. A recent study has treated the case of the translational dynamics of surface probes (M.M.G. Krishna, R. Das, N. Periasamy and R. Nityananda, J. Chem. Phys., 112 (2000) 8502-8514) using Monte Carlo and theoretical methods. Here we extend the application of the methodologies to apply the case of rotational dynamics of surface probes. The corresponding fluorescence anisotropy decays were obtained using the Monte Carlo simulation methods for the two cases: surface probes undergoing rotational dynamics on a plane and on a sphere. The results were consistent with the theoretical equations which show that Monte Carlo methods can be used to simulate the surface diffusion problems. The anisotropy decay for the rotational diffusion of a molecule on a planar surface is single exponential and the residual anisotropy is zero. However, residual anisotropy is finite for the case of rotational diffusion on a sphere because of the spatial averaging of the anisotropy function. The rotational correlation time in both the cases is (4Drot)(-1) with Drot being the rotational diffusion coefficient. Rotational dynamics of a surface bound dye in a single giant liposome and in sonicated vesicles were studied and the results were explained according to the theoretical equations. A fast component of fluorescence depolarization was also observed for sonicated vesicles which was interpreted as wobbling-in-cylinder dynamics of the surface-bound dye.     

Effects of unsaturation and curvature on the transverse distribution of intramolecular dynamics of dipyrenyl lipids.

The roles of acyl chain unsaturation and curvature in the excimer formation efficiency (EFE) of site-specific conjugated pyrene molecules in lipid membranes have been investigated by steady-state and time-resolved fluorescence spectroscopy. Six 1-2-(pyrenyl-n-acyl)-phosphatidylcholine (dipy(n)PC) probes, with pyrenyl chains of varying methylene units n from 4 to 14 carbons, were incorporated separately into dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE) lipid membranes at 0.1 mol%. Both the excimer-to-monomer fluorescence intensity ratio and association-to-dissociation rate constant ratio of conjugated pyrenes were used to quantify EFE. At all temperatures (T = 0-30 degrees C) and for n = 4 and 6, the EFE for DOPE was always smaller than EFE for DOPC. At T < 10 degrees C (where DOPE and DOPC are in the liquid crystalline L alpha phase) and for n > 8, the EFE for curvature frustrated DOPE was significantly greater than EFE for nonfrustrated DOPC (control), and the difference increased gradually with n. At T> 18 degrees C (where DOPE is in the inverted hexagonal H(II) phase and DOPC is in the L alpha phase) and for n > 8, EFE for the curvature-relaxed DOPE was again smaller than the EFE for DOPC control. The contributions of splay conformation and internal dynamics of pyrenyl chains to EFE were examined separately using a lattice model. Our results suggest that i) the cis double bonds of the host lipid matrix strongly perturb both the conformation and dynamics of conjugated pyrenes at the specific location around n = 8, and ii) the lateral stress at the upper part (n < 8) of the curvature frustrated bilayer membranes (DOPE) may be significantly relaxed once the membrane surface adopts a favorable negative interfacial curvature.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and beta-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol beta-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n=14-22 is the even number of acyl chain carbons) was studied at 30 degrees C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Kucerka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n=18-22 similarly. beta-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 A(2) and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and beta-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.     

Fast laser-induced solute release from liposomes sensitized with photochromic lipid: effects of temperature, lipid host, and sensitizer concentration.

Liposomes of gel-phase phospholipid have been prepared containing a photochromic lipid sensitizer. A fast UV laser pulse isomerizes the sensitizer destabilizing the lipid bilayer structure and causing release of trapped solute. The kinetics of solute release have been investigated as a function of host lipid chain length, sensitizer concentration, and temperature, and the limits of liposome stability have been established. At low concentrations of sensitizer, pulsed laser irradiation induces some solute release when continuous UV illumination is ineffective. Although rates of solute release usually increase with temperature, at low sensitizer concentration in a rigid host, leakage at first increases but then decreases rapidly above a threshold temperature. The results presented are relevant to the design of photostimulated drug delivery systems and to potential applications of photosensitive liposomes as caging agents for biological effectors.     

Specific binding of avidin to biotin containing lipid lamella surfaces studied with monolayers and liposomes

The interaction of avidin (from egg white) with phospholipid (monolayer and bilayer) model membranes containing biotin-conjugated phospholipids has been studied. In the first part, using surface sensitive techniques (ellipsometry and surface plasmon resonance) we demonstrated that the nonspecific adsorption of avidin to phospholipid lamella could be abolished by adding an amount of Ca²⁺, Mg²⁺, or Ba²⁺ that led to an electrostatic interaction. The specific binding of avidin to lipid mixtures containing biotin-conjugated phospholipids was obviously composition dependent. The ratio 1:12 of a B-DPPE/DPPE mixture was found to be the optimum molar ratio. When we compared the results from the surface sensitive techniques with those from the electron micrographs of a two dimensional crystal of avidin (obtained in our laboratory), the optimum ratio was found to be determined by the effect of lateral steric hindrance. In the second part, we observed the pattern of the layers of fluorescently labeled phospholipid and adsorbed proteins with a home-made micro fluorescence film balance. The fluorescence images showed that avidin was preferentially bound to the receptors that were in the fluid domains. Further, with a sensitive fluoresence assay method, the effect of the phase behavior of liposomes on the specific binding of avidin was measured. This showed that avidin interacted with biotin-lipid more weakly in the gel state liposome than in the liquid state liposome. The major conclusion was that the binding of avidin to a membrane bound model receptor was significantly restricted by two factors: one was the lateral steric hindrance and the other was the fluidity of the model membrane.Abbreviations B-DPPE Biotinyl dipalmitoylphosphatidyl ethanolamine - B-DMPE Biotinyl dimyristoylphosphatidyl ethanolamine - BNHS d-biotin-N-hydroxysuccinimide ester - DMPA dimyristoylphosphatidyl acid - DMPC dimyristoylphosphatidyl choline - DMPS dimyristoylphosphatidyl serine - DOPC dioleoylphosphatidyl choline - DPPC dipalmitoylphosphatidyl choline - DPPE dipalmitoylphosphatidyl ethanolamine - FITC fluorescein isothiocyanate - RDB-DOPE N(Lissamine rhodamine B sulfonyl) dioleoyl phosphatidylethanolamine - SPR surface plasmon resonance Correspondence to: S. F. Sui     

Thermodynamics and dynamics of the formation of spherical lipid vesicles

We propose a free energy expression accounting for the formation of spherical vesicles from planar lipid membranes and derive a Fokker–Planck equation for the probability distribution describing the dynamics of vesicle formation. We find that formation may occur as an activated process for small membranes and as a transport process for sufficiently large membranes. We give explicit expressions for the transition rates and the characteristic time of vesicle formation in terms of the relevant physical parameters.     

Charged lipid vesicles: effects of salts on bending rigidity, stability, and size

The swelling behavior of charged phospholipids in pure water is completely different from that of neutral or isoelectric phospholipids. It was therefore suggested in the past that, instead of multilamellar phases, vesicles represent the stable structures of charged lipids in excess water. In this article, we show that this might indeed be the case for dioleoylphosphatidylglycerol and even for dioleoylphosphatidylcholine in certain salts. The size of the vesicles formed by these lipids depends on the phospholipid concentration in a way that has been predicted in the literature for vesicles of which the curvature energy is compensated for by translational entropy and a renormalization of the bending moduli (entropic stabilization). Self-consistent field calculations on charged bilayers show that the mean bending modulus kc and the Gaussian bending modulus k have opposite sign and /k/>kc, especially at low ionic strength. This has the implication that the energy needed to curve the bilayer into a closed vesicle Eves=4pi(2kc+k) is much less than one would expect based on the value of kc alone. As a result, Eves can relatively easily be entropically compensated. The radii of vesicles that are stabilized by entropy are expected to depend on the membrane persistence length and thus on kc. Experiments in which the vesicle size is studied as a function of the salt and the salt concentration correlate well with self-consistent field predictions of kc as a function of ionic strength.     

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

Influences of membrane curvature in lipid hexagonal phases studied by deuterium NMR spectroscopy

The presence of reversed hexagonal phase, HII, favoring lipids in membranes has been proposed to be significant in various biological processes. Therefore an understanding of the HII phase and the transition from the lamellar to hexagonal phase is of importance. We have applied deuterium NMR spectroscopy to study the bilayer and reversed hexagonal phases of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamin e. The difference in packing between the HII and L alpha phases leads to smaller segmental order parameters in the former case. Since the order profiles are sensitive to the geometry of the aggregates, they can be used to extract structural information about the phases. We present a new means of calculating the radius of curvature, R1, for the HII phase from 2H NMR data. This method gives a value of R1 = 18.1 A, which is in agreement with current understanding of the structure of the HII phase and with x-ray diffraction data.     

Membrane dynamics as seen by fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-to-lipid ratio

The roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA(-)/desD(-)) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH(2) stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester C&z.dbnd6;O vibration. The contributions to the membrane dynamics of an ab ovo present 'structural' lipid disorder due to the protein-lipid interactions and of a thermally induced 'dynamic' lipid disorder could be distinguished.     

Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations

Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a palmitoyl-oleoyl-phosphatidylcholine bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows us to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four-state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid headgroups on both leaflets, and a second intermediate (prepore), where a polar chain across the bilayer is formed by 3-4 lipid headgroups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 ± 0.15 nm is seen, in favorable agreement with conductance measurements and electrooptical experiments of lipid vesicles.     

Comparison of dynamics of lecithin liposomes and brain total lipid liposomes with synaptosomal membranes. An EPR spectroscopy study.

Dynamics and/or order of the hydrophobic part of phosphatidylcholine (PC) liposomes and rat brain total lipid (TL) liposomes and synaptosomes were studied and compared by EPR spectroscopy using the spin probes 5 or 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The dynamics and/or order of the hydrophobic part of TL liposomes or synaptosomes were similar but differed largely from those of PC liposomes. The dynamics of the hydrophobic part of the liposomes decreased gradually with the increasing TL/PC ratio in the sample. To obtain in TL liposomes or synaptosomes the same EPR spectrum parameters as in PC liposomes at 37 degrees C, the formers have to be heated to temperatures of approximately 50-60 degrees C. The dynamics and/or order of the hydrophobic part of lecithin liposomes at 5-10 degrees C were comparable with those of TL liposomes or synaptosomes at 37 degrees C. The results emphasize the role of the lipid composition in studies concerning drug-lipid and protein-lipid interactions in model and biological membranes.     

Molecular dynamics simulation of unsaturated lipid bilayers at low hydration: parameterization and comparison with diffraction studies.

A potential energy function for unsaturated hydrocarbons is proposed and is shown to agree well with experiment, using molecular dynamics simulations of a water/octene interface and a dioleoyl phosphatidylcholine (DOPC) bilayer. The simulation results verify most of the assumptions used in interpreting the DOPC experiments, but suggest a few that should be reconsidered. Comparisons with recent results of a simulation of a dipalmitoyl phosphatidylcholine (DPPC) lipid bilayer show that disorder is comparable, even though the temperature, hydration level, and surface area/lipid for DOPC are lower. These observations highlight the dramatic effects of unsaturation on bilayer structure.     

Effects of normal alcohols and isoflurane on lipid headgroup dynamics in nicotinic acetylcholine receptor-rich lipid vesicles

The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.