Quantifying the effects of deuterium substitution on phospholipid mixing in bilayer membranes. A nearest-neighbor recognition investigation

Nearest-neighbor recognition measurements have established that the effects of deuterium substitution on phospholipid mixing are exceedingly small. Thus, the mixing behavior of an exchangeable phospholipid bearing two stearoyl chains with a homologue containing two myristoyl chains in gel-fluid bilayers, fluid bilayers, cholesterol-rich fluid bilayers, and gel-fluid bilayers that have been enriched with cholesterol correspond to a difference in the free energy of mixing that is less than 2.2 cal/mol of hydrogen in all cases. These findings provide the strongest evidence to date in support of the use of deuterated phospholipids as "nonperturbing" probes for structural and dynamic studies of bilayer membranes.     

A quantitative assessment of the influence of permanent kinks on the mixing behavior of phospholipids in cholesterol-rich bilayers

The mixing behavior of a phospholipid containing a cis-cyclopropyl moiety (1) with one that contains two myristoyl groups (3a) has been investigated in fluid bilayers via the nearest-neighbor recognition (NNR) method. In the absence of cholesterol, these lipids mix ideally. In the presence of cholesterol, they show a modest preference for homo-phospholipid association. A trans-form of 1 (i.e., 2) was found to have similar behavior, except that the influence of cholesterol in promoting homo-phospholipid association was greater. Similar results have been found in membranes in which 3a is replaced with a phospholipid bearing two palmitoyol chains (3b). In this case, the effect of the kink is approximately twice as great. The implications of these findings, with respect to the "trans-fatty acid debate", are briefly discussed     

Transbilayer complementarity of phospholipids. A look beyond the fluid mosaic model

Lipid-lipid interactions across a phospholipid bilayer were probed by measuring the nearest-neighbor preferences of exchangeable phospholipid monomers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) in the presence of nonexchangeable DMPE- or DSPE-based dimers. Each of these permanent dimers promoted homophospholipid association to the same extent, whereas the corresponding nonexchangeable monomers were without effect. These results support a model in which the longer phospholipids in one monolayer leaflet preferentially associate with shorter ones in the adjoining monolayer. Such transbilayer complementarity is likely to play an important role in stabilizing biological membranes and also in promoting a compositional interdependence of their two lipid leaflets.     

Modification of Lipid Bilayer Structure by Diacylglycerol: A Comparative Study of Diacylglycerol and Cholesterol

Diacylglycerols (DAGs) are important second messengers in biomembranes, and they can activate protein kinase C and many other enzymes and receptors. However, their interactions with cholesterol and other lipids have not been previously studied using molecular dynamics (MD) simulation. In this study, nine independent atomistic MD simulations were performed to specifically investigate the interactions between di16:0DAG, 16:0,18:1-phosphatidylcholine (POPC), and cholesterol. Despite of their substantial differences in chemical structure, DAG and cholesterol produce some very similar effects in POPC bilayers: increasing acyl chain order and bilayer thickness, reducing volume-per-lipid, and decreasing lateral diffusion of molecules. More significantly, DAG also produces a strong "condensing effect" in PC bilayers. In comparison, cholesterol is more effective than DAG in producing the above effects. The driving force for the condensing effect is their molecular shape: DAG and cholesterol both have small polar headgroups and large hydrophobic bodies. In a lipid bilayer, in order to avoid the unfavorable exposure of their hydrophobic parts to water, neighboring phospholipid headgroups move toward cholesterol or DAG to provide cover. Thus, seemingly complex interactions between DAG, cholesterol and phospholipid can be clearly explained using the Umbrella Model. Our simulations confirmed the hypothesis that DAG increases the spacing between phospholipid headgroups, which is important for activating protein kinase C and other enzymes. Interestingly, our simulations also show that the conventional wisdom that the spacing created by a DAG is directly above the DAG molecule is incorrect; instead, the largest spacing usually occurs between the first and the second nearest-neighbor PC headgroups from a DAG, due to the umbrella effect.     

Transbilayer complementarity of phospholipids. Proof of principle

A series of nearest-neighbor recognition (NNR) experiments have been carried out, which provide a rigorous test of the existence of transbilayer complementarity of phospholipids, that is, the ability of phospholipids to select complementary phospholipids from an adjoining monolayer as nearest neighbors. The application of this test to membranes derived from exchangeable phospholipids bearing myristoyl groups (A), stearoyl groups (B), and one stearoyl and one n-dodecyl group (C) in the presence of analogous nonexchangeable templates made from A', B' and C' provides compelling evidence for such complementarity in the physiologically relevant fluid phase.     

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

We report the utilization of magnetically aligned phospholipid bilayers (bicelles) to study the effects of cholesterol in phospholipid bilayers for both chain perdeuterated DMPC and partially deuterated alpha-[2,2,3,4,4,6-d(6)]-cholesterol using (2)H solid-state NMR spectroscopy. The quadrupolar splittings at 40 degrees C were 25.5 and 37.7 kHz, respectively, for the 2,4-(2)H(eq) and 2,4-(2)H(ax) deuterons when the bilayer normal of the discs was aligned perpendicular to the static magnetic field. The quadrupolar splittings were doubled when Yb(3+) ions were added to flip the bicelles 90 degrees such that the bilayer normal was colinear with the magnetic field. The results suggest that cholesterol is incorporated into the bicelle discs. For chain perdeuterated DMPC-d(54), incorporated into DMPC-DHPC bicelle discs, the individual quadrupolar splittings of the methylene and methyl groups doubled on going from the perpendicular to the parallel alignment. Also, the presence of cholesterol increased the overall ordering of the acyl chains of the phospholipids. S(CD) (i) calculations were extracted directly from the (2)H quadrupolar splittings of the chain perdeuterated DMPC. The order parameter, S(CD) (i), calculations clearly indicated an overall degree of ordering of the acyl chains in the presence of cholesterol. We also noted a disordering effect at higher temperatures. This study demonstrates the ease with which (2)H order parameters can be calculated utilizing magnetically aligned phospholipid bilayers when compared with randomly dispersed membrane samples.     

Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.     

Lipid Raft Formation: Key Role of Polyunsaturated Phospholipids

The forces that drive lipid raft formation are poorly understood. To date, most of the attention has focused on attractive interactions between cholesterol and high‐melting lipids. Remarkably little attention has been paid to repulsive forces. Here, we show that repulsive interactions between an exchangeable mimic of cholesterol and an exchangeable mimic of a low‐melting phospholipid in liquid‐disordered bilayers can be much stronger than the attractive forces between this same sterol and an exchangeable mimic of a high‐melting phospholipid in liquid‐ordered bilayers. We conclude that polyunsaturated phospholipids have been largely overlooked as major players in lipid raft formation. This knowledge should stimulate considerable interest in controlling the levels of polyunsaturated phospholipids for the proper functioning of cell membranes.     

Asymmetric or symmetric bilayer formation during oblique drop impact depends on rheological properties of saturated and unsaturated lipid monolayers

Bilayer structures are formed by approaching two liquid surfaces with phospholipid monolayers, which are brought into contact by oblique drop impact on a liquid surface. Asymmetric bilayers can be produced by the coupling of drop and target monolayers. In contrast, symmetric bilayers or multilayers are formed by collapse of the compressed target monolayer. We show that under all studied conditions bilayer/multilayer synthesis takes place. The experimental conditions for the synthesis of asymmetric or symmetric bilayers are described quantitatively in terms of the surface rheological (surface elasticity and dilational viscosity) and the hydrodynamical parameters (Weber number and impact angle). The composition and mechanical properties of the phospholipid monolayers strongly influences the patterns of drop impact and the bilayer/multilayer formation. Cholesterol stiffens unsaturated phospholipid monolayers and fluidifies saturated monolayers. All monolayers form asymmetric vesicle-like structures, which are stable in the aqueous medium. Additionally, unsaturated phospholipid monolayers without cholesterol form symmetric vesicles by folding parts of the target monolayer. Sufficient presence of cholesterol in unsaturated phospholipid monolayers inhibits the folding of the target monolayer and the subsequent formation of symmetric bilayers. The rheological properties of saturated and unsaturated phospholipid monolayers and their mixtures with cholesterol are discussed. Based on drop impact results it is shown that the state of a so far undefined region in the DPPC/cholesterol phase diagram is a fluid phase.     

Micropatterned composite membranes of polymerized and fluid lipid bilayers

Micropatterned composite membranes of polymerized and fluid lipid bilayers were constructed on solid substrates. Lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and subsequent removal of nonreacted monomers by a detergent solution (0.1 M sodium dodecyl sulfate (SDS)) yielded a patterned polymeric bilayer matrix on the substrate. Fluid lipid bilayers of phosphatidylcholine from egg yolk (egg-PC) were incorporated into the lipid-free wells surrounded by the polymeric bilayers through the process of fusion and reorganization of suspended small unilamellar vesicles. Spatial distribution of the fluid bilayers in the patterned bilayer depended on the degree of photopolymerization that in turn could be modulated by varying the applied UV irradiation dose. The polymeric bilayer domains blocked lateral diffusion of the fluid lipid bilayers and confined them in the defined areas (corrals), if the polymerization was conducted with a sufficiently large UV dose. On the other hand, lipid molecules of the fluid bilayers penetrated into the polymeric bilayer domains, if the UV dose was relatively small. A direct correlation was observed between the applied UV dose and the lateral diffusion coefficient of fluorescent marker molecules in the fluid bilayers embedded within the polymeric bilayer domains. Artificial control of lateral diffusion by polymeric bilayers may lead to the creation of complex and versatile biomimetic model membrane arrays.     

Ethanol-induced reorganization of the liquid-ordered phase: enhancement of cholesterol-phospholipid association

This paper records what is believed to be the first evidence for the reorganization of the liquid-ordered phase by ethanol. Specifically, ethanol has been found to significantly enhance sterol-phospholipid association in liquid-ordered bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) plus cholesterol and also 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) plus cholesterol. The evidence for such reorganization comes from a series of nearest-neighbor recognition (NNR) experiments that have been carried out, where low concentrations of equilibrating lipid dimers (i.e., "reporter molecules") have been used to detect changes in the phase composition of host membranes made from varying mixtures of DPPC/cholesterol, and also DSPC/cholesterol, in the presence and in the absence of ethanol. These findings have important biological implications, which are briefly discussed.     

Probing the hydration of lipid bilayers using a solvent isotope effect on phospholipid mixing

Nearest-neighbor recognition experiments, which have been carried out using exchangeable dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,and 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, indicate that replacement of H2O by D2O can significantly influence phospholipid mixing, but only in bilayers that are saturated and devoid of cholesterol. These findings, together with those of previous electron spin resonance spin-labeling studies,indicate that mammalian membranes, which are rich in cholesterol and unsaturated phospholipids, are ideal hydrophobic barriers.     

Detection of unusual lipid mixing in cholesterol-rich phospholipid bilayers: the long and the short of it

Nearest-neighbor recognition studies have revealed that favored sterol-phospholipid associations can be reversed in a fluid bilayer that contains relatively long (high melting) and short (low melting) phospholipids, when the sterol content is sufficiently high; that is, like-lipids now become favored nearest-neighbors. A possible origin of this effect is briefly discussed.     

Elastic deformation of membrane bilayers probed by deuterium NMR relaxation

In deuterium ((2)H) NMR spectroscopy of fluid lipid bilayers, the average structure is manifested in the segmental order parameters (S(CD)) of the flexible molecules. The corresponding spin-lattice relaxation rates (R(1Z) depend on both the amplitudes and the rates of the segmental fluctuations, and indicate the types of lipid motions. By combining (2)H NMR order parameter measurements with relaxation studies, we have obtained a more comprehensive picture of lipids in the liquid-crystalline (L(alpha)) state than formerly possible. Our data suggest that a lipid bilayer constitutes an ordered fluid, in which the phospholipids are grafted to the aqueous interface via their polar headgroups, whereas the fatty acyl chains are in effect liquid hydrocarbon. Studies of (2)H-labeled saturated lipids indicate their R(1Z) rates and S(CD) order parameters are correlated by a model-free, square-law functional dependence, signifying the presence of relatively slow bilayer fluctuations. A new composite membrane deformation model explains simultaneously the frequency (magnetic field) dependence and the angular anisotropy of the relaxation. The results imply the R(1Z) rates are due to a broad spectrum of 3-D collective bilayer excitations, together with effective axial rotations of the lipids. For the first time, NMR relaxation studies show that the viscoelastic properties of membrane lipids at megahertz frequencies are modulated by the lipid acyl length (bilayer thickness), polar headgroups (bilayer interfacial area), inclusion of a nonionic detergent (C(12)E(8)), and the presence of cholesterol, leading to a range of bilayer softness. Our findings imply the concept of elastic deformation is relevant on lengths approaching the bilayer thickness and less (the mesoscopic scale), and suggest that application of combined R(1Z) and S(CD) studies of phospholipids can be used as a simple membrane elastometer. Heuristic estimates of the bilayer bending rigidity kappa and the area elastic modulus K(a) enable comparison to other biophysical studies, involving macroscopic deformation of thin membrane lipid films. Finally, the bilayer softness may be correlated with the lipid diversity of biomembranes, for example, with regard to membrane curvature, repulsive interactions between bilayers, and lipid-protein interactions.     

Lipid bilayer deposition and patterning via air bubble collapse

We report a new method for forming patterned lipid bilayers on solid substrates. In bubble collapse deposition (BCD), an air bubble is first "inked" with a monolayer of phospholipid molecules and then touched to the surface of a thermally oxidized silicon wafer and the air is slowly withdrawn. As the bubble shrinks, the lipid monolayer pressure increases. Once the monolayer exceeds the collapse pressure, it folds back on itself, depositing a stable lipid bilayer on the surface. These bilayer disks have lateral diffusion coefficients consistent with high quality supported bilayers. By sequentially depositing bilayers in overlapping areas, fluid connections between bilayers of different compositions are formed. Performing vesicle rupture on the open substrate surrounding this bilayer patch results in a fluid but spatially isolated bilayer. Very little intermixing was observed between the vesicle rupture and bubble-deposited bilayers.     

A statistical mechanical model of cholesterol/phospholipid mixtures: linking condensed complexes, superlattices, and the phase diagram

Despite extensive studies for nearly three decades, lateral distribution of molecules in cholesterol/phospholipid bilayers remains elusive. Here we present a statistical mechanical model of cholesterol/phospholipid mixtures that is able to rationalize almost every critical mole fraction (X(cr)) value previously reported for sterol superlattice formation as well as the observed biphasic changes in membrane properties at X(cr). This model is able to explain how cholesterol superlattices and cholesterol/phospholipid condensed complexes are interrelated. It gives a more detailed characterization of the LG(I)region (a broader region than the liquid disordered-liquid ordered mixed-phase region), which is considered to be a sludgelike mixture of fluid phase and aggregates of rigid clusters. A rigid cluster is formed by a cholesterol molecule and phospholipid molecules that are condensed to the cholesterol. Rigid clusters of similar size tend to form aggregates, in which cholesterol molecules are regularly distributed into superlattices. According to this model, the extent and type of sterol superlattices, thus the lateral distribution of the entire membrane, should vary with cholesterol mole fraction in a delicate, predictable, and nonmonotonic manner, which should have profound functional implications.     

SHORT-RANGE MOLECULAR INTERACTIONS AND STABILITY OF AMPHIPHILE BILAYER

A new approach is proposed for the study of short-range molecular interactions between first neighboring molecules in amphiphile bilayers by means of experimentally determined dependencies of the bilayer mean lifetime on the surfactant concentration and of the critical concentration for bilayer formation on the temperature. Short-range molecular interactions are demonstrated by the parameters binding energy Q of an amphiphile molecule in the bilayer and specific line energy γ of the nucleus hole. These parameters are calculated from the experimental dependencies by using the hole-nucleation theory [D. Kashchiev and D. Exerowa, J. Colloid Interface Sci., 77, 501 (1980)] describing the stability of amphiphile bilayers. It is shown the decisive role of the short-range molecular interactions for the bilayer stability and the occurrence of first-order phase transition in phospholipid foam bilayers. The values of Q and γ are calculated for various amphiphile bilayers: anionic and nonionic surfactants, phospholipids and their natural mixtures.     

The antimalarial agent halofantrine perturbs phosphatidylcholine and phosphatidylethanolamine bilayers: a differential scanning calorimetric study.

The interaction of halofantrine with phosphatidylcholine and phosphatidylethanolamine bilayers has been investigated by differential scanning calorimetry. Halofantrine caused a broadening of the gel to liquid crystalline phase transition endotherm of the phosphatidylcholines. A decrease in the transition temperature Tm and enthalpy (delta H) of transition was also observed. This varied with the chain length of the phospholipid and was more pronounced with short chain members. Halofantrine-induced changes to the thermotropic characteristics of dipalmitoylphosphatidylcholine (DPPC)/cholesterol bilayers suggested that the penetration of halofantrine into the bilayer was diminished in the presence of cholesterol. A more complex calorimetric profile was observed in the interaction of halofantrine with phosphatidylethanolamines and the results suggested that halofantrine did not disrupt the cooperativity of the phosphatidylethanolamine bilayers to the same extent as that observed with the phosphatidylcholines. Halofantrine caused significant perturbation of phospholipids and this property might have an important bearing on its pharmacodynamic effects.     

Structural studies of the monolayers and bilayers formed by a novel cholesterol-phospholipid chimera

Langmuir isotherm, neutron reflectivity, and small angle neutron scattering studies have been conducted to characterize the monolayers and vesicular bilayers formed by a novel chimeric phospholipid, ChemPPC, that incorporates a cholesteryl moeity and a C-16 aliphatic chain, each covalently linked via a glycerol backbone to phosphatidylcholine. The structures of the ChemPPC monolayers and bilayers are compared against those formed from pure dipalmitoylphoshatidylcholine (DPPC) and those formed from a 60:40 mol % mixture of DPPC and cholesterol. In accord with previous findings showing that very similar macroscopic properties were exhibited by ChemPPC and 60:40 mol % DPPC/cholesterol vesicles, it is found here that the chimeric lipid and lipid/sterol mixture have very similar monolayer structures (each having a monolayer thickness of ～26 ?), and they also form vesicles with similar lamellar structure, each having a bilayer thickness of ～50 ? and exhibiting a repeat spacing of ～65 ?. The interfacial area of ChemPPC, however, is around 10 ?(2) greater than that of the combined DPPC/cholesterol unit in the mixed lipid monolayer (viz., 57 ± 1 vs 46 ± 1 ?(2), at 35 mN·m(-1)), and this difference in area is attributed to the succinyl linkage which joins the ChemPPC steroid and glyceryl moieties. The larger area of the ChemPPC is reflected in a slightly thicker monolayer solvent distribution width (9.5 vs 9 ? for the DPPC/cholesterol system) and by a marginal increase in the level of lipid headgroup hydration (16 vs 13 H(2)O per lipid, at 35 mN·m(-1)).     

Cholesterol-phospholipid complexation in fluid bilayers as evidenced by nearest-neighbor recognition measurements

Nearest-neighbor recognition experiments have been carried out using varying ratios of exchangeable dimer analogs of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol and 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol in cholesterol-rich unilamellar vesicles at 60 degrees C. Equilibrium dimer distributions that were obtained support a structural model of the liquid-ordered bilayer in which free cholesterol and the longer-chain phospholipid homodimer are in equilibrium with a complex of unique stoichiometry, where one cholesterol molecule combines with two of the long-chain phospholipid homodimers. In this model, the mixing of the short-chain phospholipids with the uncomplexed long-chain phospholipids is ideal, and the complexed dimers are shielded from the disulfide exchange reaction.