Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.     

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.     

Influence of vitamin E on phosphatidylethanolamine lipid polymorphism

The effect of vitamin E, in its major form alpha-tocopherol and its synthetic analog alpha-tocopheryl acetate, on phosphatidylethanolamine lipid polymorphism has been studied by mean of differential scanning calorimetry and 31P-nuclear magnetic resonance techniques. From the interaction of these tocopherols with dielaidoylphosphatidylethanolamine it is concluded that both molecules promote the formation of the hexagonal HII phase at temperatures lower than those of the pure phospholipid. When the tocopherols were incorporated in the saturated dimiristoylphosphatidylethanolamine, which has been shown not to undergo bilayer to hexagonal HII phase transition, up to 90 degrees C, they induce the phospholipid to partially organize in hexagonal HII phase. From our experiments it is shown that alpha-tocopherol is more effective than its analog in promoting HII phase in these systems. It is also shown that, while alpha-tocopheryl acetate does not significantly perturb the gel to liquid-crystalline phase transition of dimirystoylphosphatidylethanolamine, alpha-tocopherol does so and more than one peak appears in the calorimetric profile, indicating that lateral phase separations are taking place.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine. Effect of n-alkanols

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Modulation of the bilayer to hexagonal phase transition of phosphatidylethanolamines by acylglycerols

The effect of mono-, di- and triacylglycerols on the bilayer to hexagonal phase (HII) transition was studied by differential scanning calorimetry and 31P-NMR spectroscopy. The acylglycerols were mixed with either dielaidoylphosphatidylethanoline or with 1-palmitoyl-2-oleoylphosphatidylethanolamine. Acylglycerols of lauric, oleic and stearic acids were utilized. All of the acylglycerols lowered the bilayer to HII phase transition temperature. Diacylglycerols were much better HII phase promoters than monoacylglycerols while triacylglycerols were the most potent bilayer phase destabilizers. Fatty acid composition generally had less of an effect except for the monoacylglycerols where bilayer destabilization increased from monolaurin to monostearin to monoolein. The most marked difference in behaviour resulting from changes in the fatty acid composition of the acylglycerol occurred with tristearin. This was the only acylglycerol which decreased the bilayer to HII phase transition temperature only below a mol fraction of 0.005. Above this mol fraction, further addition of tristearin had no effect on the bilayer to HII phase transition. These results suggest that the tristearin has limited solubility in phosphatidylethanolamine.     

Thermodynamic, motional, and structural aspects of gramicidin-induced hexagonal HII phase formation in phosphatidylethanolamine

The effect of gramicidin incorporation on the thermodynamic properties of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) dispersions was investigated by differential scanning calorimetry. The results show that incorporation of gramicidin in PC systems results in a decrease of the energy content of the gel to liquid-crystalline phase transition. When incorporated in PE systems, however, the peptide does not affect the properties of the gel to liquid-crystalline phase transition with the exception that at high gramicidin concentrations the onset of the melting process is shifted to a slightly lower temperature. We therefore assume that in the lamellar gel state of PE aggregation of the peptide occurs. To get more insight into the nature of the gramicidin-PE interaction, we studied the motional and structural details of HII phase formation in gramicidin/PE systems with the use of 31P and 13C nuclear magnetic resonance (NMR) and small-angle X-ray diffraction. In agreement with earlier results [Van Echteld, C. J. A., Van Stigt, R., de Kruijff, B., Leunissen-Bijvelt, J., Verkleij, A. J., & De Gier, J. (1981) Biochim. Biophys. Acta 648, 287-291] it was shown that gramicidin incorporation lowers and broadens the bilayer to hexagonal HII phase transition in PE systems. 31P NMR chemical shift anisotropy (CSA) measurements indicated that a phase separation occurs between a gramicidin-poor lamellar phase and a gramicidin-rich HII phase. From combined CSA and spin-lattice relaxation time (T1) measurements it was suggested that in the HII phase gramicidin decreases the molecular order and increases the rate of motion of the phosphate moiety of PE. In addition, 13C NMR line width measurements indicated that the acyl chains are more disordered in the HII phase than in the lamellar phase and that a similar disorder occurs in the HII phase of the pure PE as in the gramicidin-rich HII phase. This interpretation was supported by the X-ray diffraction data, which show similar first-order repeat distances in both types of HII phase. From saturation-transfer NMR experiments in PE and gramicidin-PE mixtures it was shown that no exchange occurs between the lamellar and the HII phases in the time scale of 1-2 s, suggesting a macroscopic phase separation. Finally, we discussed the gramicidin-lipid interaction and in particular the HII phase formation by gramicidin in PE and in PC systems. It is proposed that aggregation of the peptide plays a crucial role in HII phase formation.     

A possible role of rhodopsin in maintaining bilayer structure in the photoreceptor membrane

31P-NMR measurements demonstrate that at 37 degrees C, independent of the photolytic state of the photopigment rhodopsin, the lipids in the photo-receptormembrane are almost exclusively organised in a bilayer. In strong contrast, the 31P-NMR spectra of the extracted lipids are characteristic for the hexagonal HII phase and an isotropic phase. The isotropic phase is characterised by freeze-fracture electron microscopy as particles and pits on smooth surfaces, possibly indicating inverted micelles. These results suggest a structural role for rhodopsin in maintaining the photoreceptor membrane lipids in a bilayer configuration.     

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions.

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL-HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of alpha-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents

Phosphatidylcholines were incorporated into hexagonal liquid cyrstalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecylether with D₂O. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the hexagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyl chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the ³¹P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from ³¹P-NMR. At the same time ²H-NMR exhibits a new signal from D₆₂O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems.     

Influence of retinoids on phosphatidylethanolamine lipid polymorphism.

The interaction of all-trans-retinoic acid and all-trans-retinol with dielaidoylphosphatidylethanolamine has been studied by differential scanning calorimetry and 31P-NMR spectroscopy. Increasing concentrations of all-trans-retinoic acid up to a mol fraction of 0.09 were found to induce shifts to lower temperatures of both the L beta to L alpha and L alpha to hexagonal-HII phase transitions, with a slight decrease in the enthalpy change of the transitions. At higher concentrations no further effects on the transitions were observed, and this is interpreted as indicative of a limited miscibility of retinoic acid with the phospholipid. 31P-NMR spectroscopy confirmed that the L alpha to hexagonal-HII phase transition was shifted to lower temperatures in the presence of retinoic acid. On the other hand increasing concentrations of all-trans-retinol up to a mol fraction of 0.166, induced a progressive shift of the L beta to L alpha and the L alpha to hexagonal-HII phase transitions to lower temperatures. At higher concentrations the main gel to liquid-crystalline phase transition was further displaced to lower temperatures and the lamellar to hexagonal-HII phase transition was not observed in the thermograms. 31P-NMR spectroscopy indicated that retinol was able of inducing the phospholipid to adopt the hexagonal-HII phase at temperatures even below the main gel to liquid-crystalline phase transition temperature of the pure phospholipid.     

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.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L alpha to the inverted hexagonal (HII) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L alpha phase compared to those in the presence of HII phase forming lipids. Our data furthermore imply that the HII phase is better suited to maintain high VDE activities at lower temperatures.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

Comparison of the orientational order of lipid chains in the L alpha and HII phases

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the use of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine in the study of lipid polymorphism

The change in the fluorescence properties of dioleoyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanola mine (N-NBD-PE) as an indicator of the (liquid-crystalline) bilayer-to-non-bilayer hexagonalII (HII) phase transition has been investigated. Lipid bilayer systems which are known to undergo the bilayer-to-HII phase transition on addition of Ca2+ were compared with systems which can undergo aggregation and fusion but not HII phase formation. The former included Ca2+-triggered non-bilayer transitions in cardiolipin and in phosphatidylethanolamine mixed with phosphatidylserine. The latter type of system investigated included the addition of polylysine to cardiolipin and Ca2+ to phosphatidylserine. Freeze-fracture electron microscopy was used to confirm that under the experimental conditions used, the formation of HII phase was occurring in the first type of system, but not in the second, which was stable in the bilayer state. It was found that the fluorescence intensity of N-NBD-PE (at 1 mol% of the phospholipids) increased in both types of system, irrespective of the formation of the HII phase. A dehydration at the phospholipid head group is a common feature of the formation of the HII phase, the interaction of divalent cations with phosphatidylserine and the interaction of polylysine with lipid bilayers, suggesting that this may be the feature which affects the fluorescence properties of the NBD. The finding of a fluorescence intensity increase in systems lacking HII phase involvement clearly indicates that the effect is not unique to the formation of the HII phase. Thus, while offering high sensitivity and the opportunity to follow kinetics of lipid structural changes, changes in the N-NBD-PE fluorescence properties should be interpreted with caution in the study of the bilayer-to-HII phase transition.