Hydrocarbon chain packing and molecular motion in phospholipid bilayers formed from unsaturated lecithins. Synthesis and properties of sixteen positional isomers of 1,2-dioctadecenoyl-sn-glycero-3-phosphorylcholine.

Isomers of cis-octadecenoic acid, with the double bond in each position in the hydrocarbon chain, were used to synthesize the corresponding 1,2-diacyl-sn-glycero-3phosphorylcholines (lecithins). Differential thermal analysis of the lecithins, as a function of water content, permitted evaluation of the limiting transition temperature (Tc) of each isomer. Values of Tc plotted against double bond position fell on a smooth curve with a minimum at minus 22 degrees for the dioctadec-9'-enoyl compound. The presence of a "pretransition" endotherm in differential thermal analysis of 1,2-dioctadec-15'-and 1,2-dioctadec-16'-enoyl-sn-glycero-3-phosphorylcholine implies the existence of two beta crystalline forms. This was not observed with any of the other lecithins. Enthalpy and entropy data were then obtained from differential scanning calorimetry measurements. Values of delta H were lower (7.6 plus or minus 0.1 kcal mol- minus 1) when the center of unsaturation was near the middle of the hydrocarbon chain than they were (9.6 kcal mol- minus 1) when the center of unsaturation was close to either end of the chain. However, values of delta S showed no consistent variation with double bond position. Four positional isomers of 1-octadec-cis-enoyl-2-octadecanoyl-sn-glycero-3-phosphorylcholine were synthesized. With the double bond near the middle of the chain or close to the terminal group, the Tc values of the mixed acid lecithins were higher than those of the corresponding dioctadecenoyl lecithins. 13-C nuclear magnetic resonance relaxation measurements were used to obtain information about chain motion of selected 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines at a temperature (52 degrees) above the Tc values. Spin-lattice relaxation times of the resolved resonances indicated that location of double bonds near the middle, as compared to either end, of the hydrocarbon chain favors enhanced molecular motion along the length of the chins and especially at the terminal methyl end. In the gel state, the minimum interaction potential energy of hydrocarbon chains in bilayers formed from dioctadecenoyl lipids appears to be minimized by localization of the double bond near the middle of the chains. It is suggested that in the case of homogeneous chains the double bond primarily affects the cooperativity of interactions and has very little steric effect on van der Waals' contacts. By contrast, in bilayers of mixed lecithins, with heterogeneous chains, the steric effect may become dominant, depending on double bond position. These differences in chain packing in the gel state are promulgated beyond the phase transition to the liquid crystalline state as an enhancement of chain motion as the temperature rises above Tc.     

Effect of acyl chain mismatch on the contact mechanics of two-component phospholipid vesicle during main phase transition

It has been recently demonstrated that acyl chain mismatch of phospholipid bilayer composed of a binary lipid mixture induces component formation on the lateral plane of the bilayer [Biophys. J. 83 (2002) 1820-1883]. In this report, the contact mechanics of unilamellar vesicles composed of binary dimyristoyl-phosphatidylcholine (DMPC)/dipalmitoyl-phosphocholine (DPPC) mixtures on fused silica and amino-modified substrates is simultaneously probed by confocal-reflectance interference contrast microscopy (C-RICM) and cross-polarized light microscopy during gel to liquid crystalline transition of the lipid bilayer. C-RICM results indicate that the average degree of vesicle deformation for DMPC-rich and DPPC-rich vesicles adhering on fused silica substrate is increased by 30% and 14%, respectively, in comparison with that in pure DMPC and DPPC vesicles. Also, lateral heterogeneity induced by acyl chain mismatch increases the average magnitude of adhesion energy in DMPC-rich and DPPC-rich vesicles of all sizes by 6.4 times and 2.3 times, respectively. Similar modulation of adhesion mechanics induced by carbon chain difference is obtained on amino-modified substrate. Most importantly, the thermotropic transition of the mixed bilayer from gel (below T(m)) to fluid phase (above T(m)) further exemplifies the effect of acyl chain mismatch on the increases of degree of vesicle deformation and adhesion energy.     

The physical properties of glycosyldiacylglycerols. Calorimetric studies of a homologous series of 1,2-di-O-acyl-3-O-(beta-D-glucopyranosyl)-sn-glycerols

The polymorphic phase behavior of aqueous dispersions of a homologous series of 1,2-di-O-acyl-3-O-(beta-D-glucopyranosyl)-sn-glycerols was studied by differential scanning calorimetry. At fast heating rates, unannealed samples of these lipids exhibit a strongly energetic, lower temperature transition, which is followed by a weakly energetic, higher temperature transition. X-ray diffraction studies have enabled the assignments of these events to a lamellar gel/liquid crystalline (chain-melting) phase transition and a bilayer/nonbilayer phase transition, respectively. Whereas the values for both the temperature and enthalpy of the chain-melting phase transition increase with increasing acyl chain length, those of the bilayer/nonbilayer phase transition show almost no chain-length dependence. However, the nature of the bilayer/nonbilayer transition is affected by the length of the acyl chain. The shorter chain compounds form a nonbilayer 2-D monoclinic phase at high temperature whereas the longer chain compounds from a true inverted hexagonal (HII) phase. Our studies also show that the gel phase that is initially formed on cooling of these lipids is metastable with respect to a more stable gel phase and that prolonged annealing results in a slow conversion to the more stable phase after initial nucleation by incubation at appropriate low temperatures. The formation of these stable gel phases is shown to be markedly dependent upon the length of the acyl chains and whether they contain an odd or an even number of carbon atoms. There is also evidence to suggest that, in the case of the shorter chain compounds at least, the process may proceed via another gel-phase intermediate. In annealed samples of the shorter chain compounds, the stable gel phase converts directly to the L alpha phase upon heating, whereas annealed samples of the longer chain glycolipids convert to a metastable gel phase prior the chain melging.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

Effect of fatty acids on phase behavior of hydrated dipalmitoylphosphatidylcholine bilayer: saturated versus unsaturated fatty acids

The effect of some fatty acids on the phase behavior of hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer was investigated with special interest in possible difference between saturated and unsaturated fatty acids. The phase behavior of hydrated DPPC bilayer was followed by a differential scanning calorimetry and a Fourier transform infrared spectroscopy. The addition of palmitic acid (PA) increased the bilayer phase transition temperature with the increase of the PA content in the mixture. In addition, DPPC molecules in gel phase bilayer became more rigid in the presence of PA compared with those in the absence of PA. This effect of PA on the phase behavior of hydrated DPPC bilayer is common to other saturated fatty acids, stearic acid, myristic acid, and also to unsaturated fatty acid with trans double bond, elaidic acid. Contrary to these fatty acids, oleic acid (OA), the unsaturated fatty acid with cis double bond in the acyl chain, exhibited quite different behavior. The effect of OA on the bilayer phase transition temperature was rather small, although a slight decrease in the temperature was appreciable. Furthermore, the IR spectral results demonstrated that the perturbing effect of OA on the gel phase bilayer of DPPC was quite small. These results mean that OA does not disturb the hydrated DPPC bilayer significantly.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

Effects of hydrostatic pressure on bilayer phase behavior and dynamics of dilauroylphosphatidylcholine.

Deuterium nuclear magnetic resonance spectroscopy was used to study the thermotropic phase behavior of dilauroylphosphatidylcholine (DLPC) bilayers at pressures up to 221 MPa. Pressure was found to separate the liquid crystal to gel transition from the gel to ordered crystalline phase transition. The jump in chain order observed on cooling through the transition into the gel phase was found to be small and thus consistent with the trend in longer chain saturated diacyl phosphatidylcholines. On cooling, DLPC was observed to enter an unusual state above the transition into the gel phase. This unusual state displayed fluid-like conformational order but short transverse relaxation times. It was found to be much better pronounced and to span a broader temperature range at elevated pressure than at lower pressures. Transverse relaxation measurements of deuterons on the chain alpha-carbons revealed a substantial slowing of molecular motions within the temperature range of the unusual fluid phase. The observation of such a phase at high pressure appears to be consistent with recent reports of an unusual fluid phase, Lx, in DLPC at ambient pressure.     

Interdigitation does not affect translational diffusion of lipids in liquid crystalline bilayers.

Asymmetric phosphatidylcholine molecules with one acyl chain twice as long as the other, below their phase transition temperature, from a mixed interdigitated phase in which the longer acyl chain spans the entire bilayer. Experimental evidence in the literature suggests that, above their phase transition temperature, these molecules may still exhibit partial interdigitation, with the longer acyl chain extending partially into the opposite leaflet, and are packed more tightly than equivalent symmetric phosphatidylcholines. Using the fluorescence recovery after photobleaching technique, we have investigated the translational diffusion in multilayers of a liquid crystalline phase, asymmetric phosphatidylcholine, 1-stearoyl-2-capryl-phosphatidylcholine (C18C10PC). We used as a fluorescent probe either a phospholipid analog of the same acyl chain composition, NBD-C18C10PE, or the symmetric equivalent of the same molecular weight, N-(7-nitrobenzoxa-2,3-diazol-4-yl)-dimyristoyl-phosphatidyle thanolamine (NBD-DMPE). Translational diffusion coefficients were also determined by using both probes in multilayers of dimyristoyl-phosphatidylcholine (DMPC) and in the eutectic mixture DMPC/C18C10PC (40/60 mol). We found that in a given host lipid, NBD-C18C10PE and NBD-DMPE diffuse at the same rate, which suggests that their bilayer free area is almost identical. This result can be explained by considering that in the liquid crystalline state, the increase in molecular packing is compensated by an increase in acyl chain dynamics. This view, which is supported by literature data, clearly suggests that the acyl chain interdigitation occurring in the liquid crystalline phase is highly dynamic.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.     

The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes.

This paper describes experiments showing the importance of the fatty acid chain length on the barrier properties of liposomal bilayers, prepared from saturated lecithins, under conditions of lateral phase separation. 1. Above the gel to liquid crystalline phase transition temperature, liposomes prepared from saturated lecithins with 14 or more carbon atoms per acyl chain exist as stable bilayers, which are practically impermeable to ions. 2. At temperatures well above the transition temperature dilauroyl phosphatidylcholine liposomes exhibited osmotic shrinkage, which was dependent on the ionic size of the solute used to bring about the osmotic gradient, indicating that the permeation through these less stable bilayers takes place mainly via individual diffusion of the permeating ions. 3. An enhanced release of trapped potassium from liposomes was demonstrated in the vicinity of the transition temperature. The extent of the increase, however, depended strongly on the length of the paraffin chain. 4. From measurements of the shrinkage behaviour of liposomes in the vicinity of the transition temperature it is concluded that the increased permeability decreases with increasing diameter of the permeating ion. This finding implies that the increased permeability at the transition temperature cannot be ascribed to "macroscopic" rupture of the liposomal membrane. The maximum permeability in the vicinity of the Tc is discussed in terms of probability and size distribution of statistical pore formation at the boundaries of liquid and solid domains.     

Effects of cyclosporine A on biomembranes. Vibrational spectroscopic, calorimetric and hemolysis studies.

Cyclosporine A (CSA)-dipalmitoylphosphatidylcholine (DPPC) interactions were investigated using scanning calorimetry, infrared spectroscopy, and Raman spectroscopy. CSA reduced both the temperature and the maximum heat capacity of the lipid bilayer gel-to-liquid crystalline phase transition; the relationship between the shift in transition temperature and CSA concentration indicates that the peptide does not partition ideally between DPPC gel and liquid crystalline phases. This nonideality can be accounted for by excluded volume interactions between peptide molecules. CSA exhibited a similar but much more pronounced effect on the pretransition; at concentrations of 1 mol % CSA the amplitude of the pretransition was less than 20% of its value in the pure lipid. Raman spectroscopy confirmed that the effects of CSA on the phase transitions are not accompanied by major structural alterations in either the lipid headgroup or acyl chain regions at temperatures away from the phase changes. Both infrared and Raman spectroscopic results demonstrated that CSA in the lipid bilayer exists largely in a beta-turn conformation, as expected from single crystal x-ray data; the lipid phase transition does not induce structural alterations in CSA. Although the polypeptide significantly affects DPPC model membrane bilayers, CSA neither inhibited hypotonic hemolysis nor caused erythrocyte hemolysis, in contrast to many chemical agents that are believed to act through membrane-mediated pathways. Thus, agents, such as CSA, that perturb phospholipid phase transitions do not necessarily cause functional changes in cell membranes.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. A direct comparison of the effects of cis and trans cyclopropane ring and double-bond substituents on orientational order

The hydrocarbon chain orientational order parameters of membranes of Acholeplasma laidlawii B, enriched with large quantities of fatty acids containing either a cis or a trans cyclopropane ring or a cis or trans double bond, plus small quantities of one of an isomeric series of monofluoropalmitic acids, were determined via fluorine-19 nuclear magnetic resonance spectroscopy over a range of temperatures spanning the corresponding gel to liquid-crystalline phase transitions (determined via differential scanning calorimetry). Membrane orientational order profiles in the liquid-crystalline state were generally similar, regardless of the particular fatty acid structure present, showing a region of relatively constant order preceding a region of progressively decreasing order toward the methyl terminus of the acyl chain. In the gel state, the order profiles in the presence of either a trans cyclopropane ring or trans double-bond substituent were similar and were characterized by a pronounced head to tail gradient of order at temperatures just below the lipid phase transition, while at temperatures far below the lipid phase transition this gradient was less pronounced, all chain positions showing a more uniformly high degree of orientational ordering. In the gel state, the order profiles in the presence of either a cis cyclopropane ring or a cis double-bond substituent were also similar but were highly unusual in that order first increased and only then subsequently decreased toward the acyl chain methyl terminus. In addition, the substituents in the cis configuration, whether a cyclopropane ring or a double bond, were overall more disordered in the gel state than the corresponding substituents in the trans configuration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.     

Reorganizational dynamics of multilamellar lipid bilayer assemblies using continuously scanning Fourier transform infrared spectroscopic imaging

We employ an implementation of rapid-scan Fourier transform infrared (FT-IR) microspectroscopic imaging to acquire time-resolved images for assessing the non-repetitive reorganizational dynamics of aqueous dispersions of multilamellar lipid vesicles (MLVs) derived from distearoylphosphatidylcholine (DSPC). The spatially and temporally resolved images allow direct and simultaneous determinations of various physical and chemical properties of the MLVs, including the main thermal gel to liquid crystalline phase transition, comparisons of vesicle diffusion rates in both phases and the variation in lipid bilayer packing properties between the inner and outer lamellae defining the vesicle. Specifically, in the lipid liquid crystalline phase, the inner bilayers of the MLVs are more intermolecularly ordered than the outer regions, while the intramolecular acyl chain order/disorder parameters, reflecting the overall characteristics of the fluid phase, remain uniform across the vesicle diameter. In contrast, the lipid vesicle gel phase displays no intermolecular or intramolecular dependence as a function of distance from the MLV center.     

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.     

Effects of cis and trans unsaturation on the structure of phospholipid bilayers: a high-pressure infrared spectroscopic study

In order to compare the effects of cis and trans unsaturation on the structure and packing of phospholipid bilayers, infrared spectra of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) were measured in a diamond anvil cell at 28 degrees C as a function of pressure up to 36 kbar. The infrared spectra indicate that DEPC and DOPC undergo pressure-induced liquid-crystalline to gel phase transitions at critical pressures of 0.7 and 5.2 kbar, respectively. Below their respective critical pressures, the infrared spectra of DOPC and DEPC are essentially indistinguishable, whereas above these pressures, there are very pronounced differences in the barotropic behavior of these two lipids. Specifically, at the 5.2-kbar transition in DOPC, there are significant changes in the frequencies, intensities, and widths of bands associated with the interfacial C = O groups, the olefinic CH = CH groups, and the terminal CH3 groups, whereas the corresponding bands of DEPC are, by contrast, relatively insensitive to the pressure-induced phase transition. The unusual band shape changes in DOPC are attributed to a unique packing arrangement of the oleoyl acyl chains required to accommodate the bent geometries of adjacent cis double bonds. Moreover, above 5 kbar in DEPC, well-defined correlation field splittings of the CH2 scissoring and rocking modes are observed, with magnitudes very similar to those observed at comparable pressures in saturated lipid systems. The absence of correlation field splittings of the corresponding bands of DOPC up to 36 kbar suggests that the bent oleoyl acyl chains are closely packed with all chains oriented parallel to each other.     

Structural,dynamic and mechanical properties of POPC at low cholesterol concentration studied in pressure/temperature space

We have studied the structural, dynamic and mechanical properties of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC)/cholesterol binary mixtures by small-angle X-ray scattering. Our investigations were concentrated on the biologically most relevant pressure-temperature-cholesterol regime, i.e. the liquid crystalline phase and its phase boundary to the lamellar gel phase within a cholesterol concentration up to 25 mol%. From the dependence of the transition pressure we derived a value of 19 kJ/mol for the transition enthalpy Delta H(m) of POPC in excess water. With increasing cholesterol concentration, Delta H(m) drops to about 7 kJ/mol at 20 mol% cholesterol. Time-resolved pressure-scan (p-scan) and temperature-jump (T-jump) experiments reveal that at low cholesterol content (<5-8 mol%) the fluidity and also the bilayer compressibility increase remarkably. In contrast, at concentrations between 5 and 25 mol% cholesterol the bilayer becomes again more rigid and the lipid bilayer spacing increases about 2 A. Theses changes are attributed to the onset of phase separation between liquid disordered and liquid ordered phases. The fluid-fluid miscibility gap for this mono-unsaturated lecithin species is strongly enlarged compared with saturated lecithins.     

Effects of anesthetic tetradecenols on phosphatidylcholine phase transitions. Implications for the mechanism of the bilayer pretransition.

The effects of cis- and trans-9,10-tetradecenols on the phase transitions of dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylcholines were investigated using high sensitivity scanning calorimetry and Raman spectroscopy. Both alcohols lowered the gel to liquid crystalline phase transition temperatures for all three phosphatidylcholines, with cis-tetradecenol showing a considerably greater effect than trans-tetradecenol in each case. While both alcohols increased the temperature of the dimyristoylphosphatidylcholine pretransition, and decreased the temperature of the distearoylphosphatidylcholine pretransition, cis-tetradecenol lowered the temperature of the dipalmitoylphosphatidylcholine pretransition, while trans-tetradecenol dramatically raised the pretransition temperature. These results are interpreted in terms of the reduction in gel (L beta) phase chain tilt and changes in the ease of acyl chain trans-gauche isomerization which are introduced by the alcohols, and the consequent effects of these changes on the pretransition and the gel to liquid crystalline phase transition. The data clearly show that caution is necessary in applying information on lipid-anesthetic interactions obtained from model membranes to the problem of clinical anesthesia, since qualitatively different results may be obtained when lipids of differing acyl chain lengths are employed. Superficial interpretation of such data might lead to erroneous conclusions.