Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions

The lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transitions of aqueous dispersions of a number of synthetic phosphatidylethanolamines containing linear saturated, branched chain, and alicyclic fatty acyl chains of varying length were studied by differential scanning calorimetry, 31P nuclear magnetic resonance spectroscopy, and X-ray diffraction. For any given homologous series of phosphatidylethanolamines containing a single chemical class of fatty acids, the lamellar gel/liquid-crystalline phase transition temperature increases and the lamellar liquid-crystalline/reversed hexagonal phase transition temperature decreases with increases in hydrocarbon chain length. For a series of phosphatidylethanolamines of the same hydrocarbon chain length but with different chemical structures, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary markedly and in the same direction. In particular, at comparable effective hydrocarbon chain lengths, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary in parallel, such that the temperature difference between these two phase transitions is nearly constant. Moreover, at comparable effective acyl chain lengths, the d spacings of the lamellar liquid-crystalline phases and of the inverted hexagonal phases are all similar, implying that the thickness of the phosphatidylethanolamine bilayers at the onset of the lamellar liquid-crystalline/reversed hexagonal phase transition and the diameter of the water-filled cylinders formed at the completion of this phase transition are comparable and independent of the chemical structure of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral diffusion study of excimer-forming lipids in lamellar to inverted hexagonal phase transition of unsaturated phosphatidylethanolamine

Using multi-frequency cross-correlation fluorometry, the monomer fluorescence lifetime of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl)phosphatidylcholine (Py-PC) was employed to determine the lateral diffusion constant (DT) of dioleoylphosphatidylethanolamine (DOPE) in both the lamellar (L alpha) and the inverted hexagonal (HII) phases. The values of DT increased with temperature in both phases. However, the rate of increase of DT declined abruptly at approximately 10-13 degrees C (L alpha -HII transition temperature), as indicated by the existence of an inflection point in the log (DT/T) vs. 1/T plot. This observation suggests that the translational motion of lipids in the HII phase is lower than that in the L alpha phase upon temperature extrapolation. Lipid perturbants, cholesterol and diacylglycerol, were found to destabilize the L alpha phase of DOPE. This was demonstrated by a down-shift of the inflection point in the log(DT/T) vs. 1/T plot in the presence of the perturbants. Both cholesterol and 1,2-dioleoyl-sn-glycerol (diolein) decreased the lateral diffusion constant in both phases. Diolein promoted the HII phase more effectively than did the cholesterol. This is explained by an intrinsic wedge-shape geometry of diolein which strongly favors the formation of inverted cylindrical packing of the lipids.     

Dependence of lipid chain and head group packing of the inverted hexagonal phase on hydration

A model which positions the hydrophobic/hydrophilic boundary in phosphatidylethanolamine lipids at the first CH₂ group in the acyl or alkyl chain is used to calculate the surface area per lipid, the mean chain and head-group dimensions and diameters of the hydrophilic tubes of the inverted hexagonal phase of didodecylphosphatidylethanolamine. The calculated surface areas compare favorably with areas obtained for the lamellar liquid crystal phase of the same lipid using the same boundary. Placement of the boundary within the lipid structure permits a determination of the maximum headgroup packing at hydration levels down to complete dehydration. The headgroup dimensions are consistent with a 5 Å diam void at the center of a hydrophilic tube at zero hydration. The calculated mean fluid chain length is ~2 Å smaller than the mean chain length of the lamellar phase at comparable levels of hydration. Comparison of the calculated mean fluid chain length and distances between hydrophobic boundaries shows that the fluid chains are interdigitated between adjacent tubes, and not interdigitated in the central space between three tubes. At low hydration the chains interdigitate in both spaces. The number of lipids packed around a tube at low hydration is only a function of the headgroup geometry, whereas at high hydration, it is a function of the number of carbon atoms in the chains.     

The role of cardiolipin concentration and acyl chain composition on mitochondrial inner membrane molecular organization and function

Cardiolipin (CL) is a key phospholipid of the mitochondria. A loss of CL content and remodeling of CL's acyl chains is observed in several pathologies. Strong shifts in CL concentration and acyl chain composition would presumably disrupt mitochondrial inner membrane biophysical organization. However, it remains unclear in the literature as to which is the key regulator of mitochondrial membrane biophysical properties. We review the literature to discriminate the effects of CL concentration and acyl chain composition on mitochondrial membrane organization. A widely applicable theme emerges across several pathologies, including cardiovascular diseases, diabetes, Barth syndrome, and neurodegenerative ailments. The loss of CL, often accompanied by increased levels of lyso-CLs, impairs mitochondrial inner membrane organization. Modest remodeling of CL acyl chains is not a major driver of impairments and only in cases of extreme remodeling is there an influence on membrane properties.     

Reversed hexagonal phase formation in lecithin-alkane-water systems with different acyl chain unsaturation and alkane length

Investigations of lipid-alkane systems are important for an understanding of the interactions between lipids and hydrophobic/amphiphilic peptides or other hydrophobic biological molecules. A study of the formation of nonlamellar phases in several phosphatidylcholine (PC)-alkane-2H2O systems has been performed. The PC molecules chosen in this work are dipalmitoyl-PC (DPPC), 1-palmitoyl-2-oleoyl-PC (POPC), dioleoyl-PC (DOPC), and dilinoleoyl-PC (DLiPC), lipids that in excess water form just a lamellar liquid-crystalline phase up to at least 90 degrees C. The addition of n-alkanes (C8-C20) to these PC-2H2O systems induces the formation of reversed hexagonal (HII) and isotropic phases. The water and dodecane concentrations required to form these phases depend on the degree of acyl chain unsaturation of the PC molecules and increase in the order DLiPC approximately DOPC less than POPC less than DPPC. The most likely explanation to this result is that the diameter of the lipid-water cylinders in the HII phase grows gradually larger with increased acyl chain saturation and more water and dodecane are consequently needed to fill the water cylinders and the void volumes between the cylinders, respectively. The ability of the alkanes to promote the formation of an HII phase is strongly chain length dependent. Although the number of alkane carbon atoms added per DOPC molecule in the DOPC-n-alkane-2H2O mixtures was kept constant, this ability decreased on going from octane to eicosane. The thermal history of a DPPC-n-dodecane-2H2O sample was important for its phase behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration and the lamellar to hexagonal II phase transition of phosphatidylethanolamine

The effects of chaotropic agents on the lamellar to hexagonal II phase transition of soy phosphatidylethanolamine were examined. Guanidine hydrochloride, urea, and NaSCN were used as chaotropic agents. In each case, the lamellar phase was stabilized by the presence of the chaotropic agent. In the case of NaSCN, the temperature of the lamellar to hexagonal phase transition of soy phosphatidylethanolamine was increased by more than 60 degrees C. Guanidine hydrochloride was capable of substantially reducing the aggregation of phosphatidylethanolamine vesicles. These data lead to a thermodynamic understanding of the lamellar to hexagonal phase transition.     

Kinetics and mechanism of the lamellar gel/lamellar liquid-crystal and lamellar/inverted hexagonal phase transition in phosphatidylethanolamine: a real-time X-ray diffraction study using synchrotron radiation

A study of the kinetics and mechanism of the thermotropic lamellar gel/lamellar liquid-crystalline and lamellar/inverted hexagonal phase transition in dihexadecylphosphatidylethanolamine (DHPE) at various hydration levels has been carried out. Measurements were made by using a real-time X-ray diffraction method at the Cornell High Energy Synchrotron Source. This represents an extension of an earlier study concerning the lamellar gel/lamellar liquid-crystalline phase transition in dipalmitoylphosphatidylcholine [Caffrey, M., & Bilderback, D. H. (1984) Biophys. J. 45, 627-631]. With DHPE, the chain-melting and the nonbilayer transitions were examined under active heating and passive cooling conditions by using a temperature jump to effect phase transformation. Measurements were made at hydration levels ranging from 0% to 60% (w/w) water, and in all cases, the transitions were found to be repeatable, be reversible, and have an upper bound on the transit times (time required to complete the transition) of less than or equal to 3 s. The shortest transit time recorded for the chain-melting and lamellar/hexagonal transitions was less than 1 s. At 8% (w/w) water, the transit times were still on the order of seconds even though the transition does not involve the intermediate L alpha phase. Note, the measured transit times are gross values incorporating the intrinsic transit time in addition to the time required to heat or cool the sample through the transition temperature range and to supply or remove the latent heat of the transition. Regardless of the direction of the transition, both appear to be two state to within the sensitivity limits of the real-time method. From simultaneous wide- and low-angle measurements at the lamellar chain-melting transition, loss of long-range order in the lamellar gel phase appears to precede the chain-melting process. On the basis of the real-time X-ray diffraction measurements, a mechanism is proposed for the lamellar/hexagonal phase transition. The mechanism does not involve large or energetically expensive molecular rearrangements, leads directly to a hexagonal lattice coplanar with the lamellar phase, incorporates facile reversibility, repeatability, and cooperativity, accounts for an observed, apparent memory in the hexagonal phase of the original lamellar phase orientation, and is consistent with the experimental observation of a predominantly two-state transition. In conjunction with the kinetic measurements, the DHPE/water phase diagram was constructed. At and above 12% (w/w) water, the thermotropic transition sequence is L beta'/L alpha/HII.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology of the intermediate stages in the lamellar to hexagonal lipid phase transition

Summary The addition of calcium to suspensions of egg phosphatidylcholine and cardiolipin converts multiwalled liposomes to the hexagonal (H_II) phase (Rand, R.P., Sengupta, S. (1972)Biochim. Biophys. Acta 255:484–492). We have studied this lamellar to hexagonal phase transition by freeze-fracture, thin-section electron microscopy, and X-ray diffraction and have morphologically characterized the intermediate stages. The first step in the transition involves the invagination and fusion of bilayers, marked by the appearance of lipidic intramembrane particles and crater-like indentations, as the large liposomes are converted to smaller flattened and elongated vesicles. The next step is the formation of tightly packed hexagonal arrays of tubules, each tubule being about 11 to 15 nm in diameter. These tubules are filled with fluid and a lipid bilayer forms the wall of each cylinder. Finally this tubular bilayer phase is converted to the hexagonal (H_II) phase, where the distance between tubes is 5.5 to 7.5 nm.     

An investigation into a cardiolipin acyl chain insertion site in cytochrome c

Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex.     

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between L alpha and HII phases

Inverted cubic and isotropic phases have been observed in phospholipid and glycolipid systems. These phases exhibit characteristic morphologies in freeze-fracture electron micrographs, isotropic 31P-NMR resonances and (in some cases) cubic X-ray diffraction patterns. It is proposed here that these phases may form from the same intermediates that are involved in lamellar/inverted hexagonal (L alpha/HII) phase transitions, and that it is possible that these cubic and isotropic phases are metastable. According to a kinetic theory of L alpha/HII phase transitions, intermediates in such transitions can form structures known as interlamellar attachments (ILAs). It is shown that ILAs should form in large numbers during L alpha/HII transitions in systems like those reported to form inverted cubic or isotropic structures. ILAs cannot readily assemble into either the HII phase or well-ordered arrays of L alpha phase bilayers, and represent a kinetic trap for intermediates in L alpha/HII transitions (although it is possible that they are marginally more stable in a thermodynamic sense than the L alpha phase in a small temperature range below TH). It is also shown that arrays of ILAs should form metastable arrays with the same morphology and isotropic 31P-NMR resonances that are observed in isotropic and inverted cubic states. In particular, under some circumstances ILAs will assemble into a structure identical to the bicontinuous inverted cubic phase previously described in monoglycerides and very similar in morphology to structures observed in phospholipid systems. Finally, since isotropic and cubic states form from ILAs, which also can mediate fusion of unilamellar vesicles, unilamellar vesicles should fuse to at least some extent under the same conditions in which multilamellar samples of the same lipid form isotropic or inverted cubic states. This correlation has been observed.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Dependence of the bilayer to hexagonal phase transition on amphiphile chain length

Several series of amphiphiles of increasing chain length were tested for their abilities to modify the L alpha-HII transition of dielaidoylphosphatidylethanolamine using differential scanning calorimetry. Acylcarnitines, alkyl sulfates, alkylsulfobetaines, and phosphatidylcholines, with chain lengths between about 6 and 12 carbon atoms, show an increasing capacity to raise the L alpha-HII phase transition temperature of phosphatidylethanolamine. This is ascribed to increased partitioning of the added amphiphile from water into the membrane as the chain length increases. Alkyl sulfates and alkyltrimethylammonium bromides have diminished capacities to raise the L alpha-HII transition temperature as the chain length is increased from 12 to 16. This is caused by an increase in the hydrophobic portion of the amphiphile leading to a change in the intrinsic radius of curvature and a decrease in the hydrocarbon packing constraints in the HII phase relative to the shorter chain amphiphiles. The L alpha-HII transition temperature of phosphatidylethanolamine with acylcarnitines of chain length 14-20 carbon atoms, alkylsulfobetaines above 14 carbon atoms, and phosphatidylcholines with acyl groups having above 10 carbon atoms is relatively insensitive to chain length. We suggest that this is caused by a balance between increasing hydrocarbon volume promoting the HII phase through decreased intrinsic radius of curvature and greater relief of hydrocarbon packing constraints vs greater intermolecular interactions favoring the more condensed L alpha phase. This latter effect is more important for amphiphiles with large headgroups which can pack more efficiently in the L alpha phase. The phosphatidylcholines show a gradual decrease in bilayer stabilization between 10 and 22 carbon atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the lamellar phase unbinding energy on the relative stability of lamellar and inverted cubic phases

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q_II) phases at temperatures between the lamellar (L_α) and inverted hexagonal (H_II) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L_α/H_II phase transitions and may form intermediate Q_II phases only after the temperature is cycled repeatedly across the L_α/H_II phase transition temperature, T_H, or when the H_II phases are cooled from T > T_H. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L_α phase bilayers, g_u, that serves to stabilize the L_α phase relative to the nonlamellar phases. The planar L_α phase bilayers must separate for a Q_II phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q_II phase from L_α at temperatures near the L_α/Q_II transition temperature (T_Q). Using g_u and elastic constant values typical of unsaturated PEs, we show that g_u is sufficient to make T_Q > T_H for the latter lipids. Such systems would display direct L_α → H_II transitions, and a Q_II phase might only form as a metastable phase upon cooling of the H_II phase. The g_u values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T_Q by only a few degrees, consistent with observations of these systems. This influence of g_u also rationalizes the effect of some aqueous solutes to increase the rate of Q_II formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T_Q and on measuring Q_II phase unit cell dimensions, respectively. In view of the effect of g_u on T_Q that we describe here, the latter method, which does not depend on the value of g_u, is preferable.     

Kinetics of the lamellar and hexagonal phase transitions in phosphatidylethanolamine. Time-resolved x-ray diffraction study using a microwave-induced temperature jump.

The kinetics of the thermotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) and L alpha/inverted hexagonal (HII) phase transitions in fully hydrated dihexadecylphosphatidylethanolamine (DHPE) have been studied. Measurements were made by using time-resolved x-ray diffraction (TRXRD) to monitor progress of the transitions. In these studies microwave energy at 2.5 GHz was used to increase the sample temperature rapidly and uniformly through the phase transition regions. The L beta'/L alpha and L alpha/HII transitions of DHPE were examined under active microwave heating and passive cooling. The transitions were found to be repeatable and reversible, and to have an upper bound on the time required to complete the transition of less than 3 s. Regardless of the direction of the transition, both phase transitions appeared to be two-state with no accumulation of intermediates to within the sensitivity limits of the TRXRD method. The rate and amplitude of the temperature jump can be controlled by regulating microwave radiation input power. A temperature jump rate of 29 degrees C/s was obtained at a final microwave power setting of 120 W. Comparisons between previously reported fluid flow (Caffrey, M. 1985. Biochemistry. 24:4826-4844) and microwave heating studies suggest that the determination of limiting transit times will require faster heating.     

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.