Pulse NMR study of phase transitions in dipalmitoyl phosphatidylcholine multilayer systems

Pretransition and main transition of aqueous dipalmitoyl phosphatidylcholine (DPPC) dispersions were investigated by pulse NMR. The second moment M2 inter of the proton absorption line shows significant changes at 42 degrees C and about 35 degree C. Over the whole investigated temperature range between 25 and 50 degree C a superposition of at least two distinct second moments assigned to different molecular regions was observed.     

Comparisons of lipid dynamics and packing in fully interdigitated monoarachidoylphosphatidylcholine and non-interdigitated dipalmitoylphosphatidylcholine bilayers: cross polarization/magic angle spinning 13C-NMR studies

13C-NMR spectra have been obtained at 50.3 MHz for monoarachidoylphosphatidylcholine (MAPC) and dipalmitoylphosphatidylcholine (DPPC) dispersions from 25 degrees C to 55 degrees C and for DPPC polycrystals at 25 degrees C using the cross polarization/magic angle spinning technique. Differential scanning calorimetric studies on DPPC and MAPC dispersions show comparable lipid phase transitions with transition temperatures at 41 degrees C and 45 degrees C, respectively, and thus enable the comparison of thermal, structural and dynamic differences between these two systems at corresponding temperatures. Conformational-dependent 13C chemical shift studies on DPPC dispersions demonstrate not only the coexistence of the tilted gel (L beta') and liquid-crystalline (L alpha) phases in the rippled gel (P beta') phase, but also the presence of an intermediate third microscopic phase as evidenced by three resolvable peaks for omega - 1 methylene carbon signals at the temperature interval between Tp and Tm. By comparing chemical shifts of MAPC in the hydrocarbon chain region with those of DPPC at similar reduced temperatures, it can be concluded that the packings are perturbed markedly in the middle segment of the fatty acyl chain during the lamellar to micellar transition. However, terminal methylene and methyl groups of interdigitated MAPC lamellae were found to be more ordered than those of non-interdigitated DPPC bilayers in the gel state. Interestingly, the terminal methyl groups of MAPC in the micelles remain to be relatively ordered; in fact, they are more ordered than the corresponding acyl chain end of DPPC in the liquid-crystalline state. Analysis of data obtained from rotating frame proton spin-lattice relaxation measurements shows a highly mobile phosphocholine headgroup, a rigid carbonyl group and an ordered hydrocarbon chain for lamellar MAPC in the interdigitated state. Furthermore, results suggest that free rotations of the glycerol C2-C3 bond within MAPC molecules may occur in the interdigitated bilayer, whereas intramolecular exchange between two conformations of the glycerol backbone in DPPC become possible at temperatures close to the pretransition temperature. Without isotope enrichment, we conclude that high-resolution solid-state 13C-NMR is indeed a useful technique which can be employed to study the packing and dynamics of phospholipids.     

Hydrostatic pressure induces hydrocarbon chain interdigitation in single-component phospholipid bilayers

By use of neutron diffraction for structural analysis, the temperature-pressure phase diagrams of several fully hydrated single-component phospholipid bilayers have been explored up to hydrostatic pressures of 2 kbars. The gel to liquid-crystalline phase transition temperature Tm increases linearly with pressure over a 10(-3)-2 kbar range in accordance with the Clausius-Clapeyron relationship giving dTm/dP values of 23.0 degrees C/kbar for 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 28.0 degrees C/kbar for 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The so-called pretransition was not observed in the isothermal pressure experiments, suggesting that no appreciable volume change occurs at this transition. These results are in good agreement with those reported using other techniques. In addition, at pressures higher than the isothermal liquid-crystalline to gel transition pressure, a new pressure-induced phase transition was observed for DPPC and DSPC in which the hydrocarbon chains from apposing monolayers become interdigitated with the chains occupying a cross-sectional area approximately equal to 5% less than in the gel phase. The temperature-pressure phase diagrams show the gel-interdigitated phase boundaries to be highly curved and the minimum pressure at which interdigitation occurs to decrease with increasing hydrocarbon chain length.     

Conformation of phosphatidylcholine in neat and cholesterol-containing liquid-crystalline bilayers. Application of a novel method.

The conformation of phosphatidylcholine in liquid-crystalline bilayers was studied with a novel, high-resolution method employing phosphatidylcholine species containing pyrenyl moieties in both acyl chains of variable length. Analysis of the intramolecular pyrene-pyrene collision data obtained for 30 such species in terms of a simple geometrical model showed that the sn-1 acyl chain penetrates, on the average, 0.84 +/- 0.11 methylene units (0.8 A) deeper into the bilayer than the sn-2 chain at 22 degrees C. A similar value was obtained at 37 degrees C. Since the penetration difference of the sn-1 and sn-2 acyl chains is inherently coupled to the conformation of the glycerol moiety, these data mean that the glycerol moiety of phosphatidylcholine is, on the average, only moderately tilted with respect to the bilayer plane in the liquid-crystalline state. This contrasts the perpendicular orientation observed previously for phosphatidylcholine crystals [Pearson, R. H., & Pascher, I. (1979) Nature 281, 499-501]. Importantly, addition of 50 mol % cholesterol, which is known to reduce dramatically the interactions between phosphatidylcholine molecules in bilayers, had only a small effect on the penetration difference of the acyl chains, strongly suggesting that the conformation of phosphatidylcholine in the liquid-crystalline state is determined largely by intramolecular, rather than intermolecular, interactions.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Orientation of specifically 13C=O labeled phosphatidylcholine multilayers from polarized attenuated total reflection FT-IR spectroscopy.

Oriented multilayers of 1-myristoyl-2(1-13C)-myristoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DMPC) and 1-palmitoyl-2(1-13C)-palmitoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DPPC) were investigated by use of attenuated total reflection infrared spectroscopy with polarized light. Experiments were performed with the aim to determine the orientation of the two ester groups in these phospholipids in the solid state and in the hydrated state at temperatures below and above the respective gel to liquid-crystalline phase transitions. Substitution of the naturally occurring 12C carbonyl carbon atom by 13C in the ester group of the sn-2 chain of DMPC and DPPC shifts the infrared absorption of the carbonyl double bond stretching vibration to lower frequency. This results in two well-resolved ester C=O bands which can be assigned unequivocally to the sn-1 and sn-2 chains as they are separated by more than 40 cm-1. The two ester CO-O single bond stretching vibrations of the molecular fragments-CH2CO-OC-are also affected and the corresponding infrared absorption band shifts by 20 cm-1 on 13C-labeling of the carbonyl carbon atom. From the dichroic ratios of the individual ester bands in 2(1-13C)DMPC and 2(1-13C)DPPC we were able to demonstrate that the sn-1 and sn-2 ester C=O groups are similarly oriented with respect to the bilayer plane, with an angle greater than or equal to 60 degrees relative to the bilayer normal. The two CO-O single bonds on the other hand have very different orientations. The CH2CO-OC fragment of the sn-1 chain is oriented along the direction of the all-trans methylene chain, whereas the same molecular segment of the sn-2 carbon chain is directed toward the bilayer plane. This orientation of the ester groups is retained in the liquid-crystalline phase. The tilt angle of the hydrocarbon all-trans chains, relative to the membrane normal, is 25 degrees in the solid state of DMPC and DPPC multibilayers. In the hydrated gel state this angle varies between 26 degrees and 30 degrees, depending on temperature. Neither the orientation of the phosphate group, nor that of the choline group varies significantly in the different physical states of these phospholipids.     

Trehalose and dry dipalmitoylphosphatidylcholine revisited

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum Tm of 24 degrees C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105-112 degrees C for dry pure DPPC and of 42 degrees C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.     

High pressure 2H-NMR study of the order and dynamics of selectively deuterated dipalmitoyl phosphatidylcholine in multilamellar aqueous dispersions.

High pressure 2H multipulse NMR techniques were used to investigate the effects of pressure on the structure and dynamics of selectively deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilamellar aqueous dispersions. The samples were deuterated on both chains at positions 2, 9, or 13. The deuterium lineshapes, the spin-lattice relaxation times, T1, and the spin-spin relaxation times, T2, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for the three deuterated DPPC samples. This pressure range permitted us to explore the phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as the Gel I (P beta), Gel II (L beta), Gel III, Gel X, and the interdigitated, Gel i, gel phase. Pressure had an ordering effect on all chain segments both in the LC phase and various high pressure gel phases as indicated by the increase in SCD bond order parameter and the first moment, M1, with pressure. Compared with the adjacent gel phases, the Gel i phase had the highest order. Also, in all gel phases the carbon-9 segment of the chains had the most restricted motions in contrast to the LC phase, where the carbon-2 segment was the most restricted. In the LC phase, T1 and T2 values for all segments decreased with pressure, indicative of the fast correlation time regime. Similarly, T1 decreased with pressure in the Gel I and the interdigitated Gel i gel phases but changed to the slow correlation time regime at the Gel i/Gel II phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

1H-NMR study of the three low temperature phases of DPPC-water systems

The three phases of dipalmitoylphosphatidylcholine-water dispersions, occurring below the main transition are studied by a moment analysis of 1H-nuclear magnetic resonance (NMR) spectra. The subtransition, recently detected by Chen, S. C., J. M. Sturtevant, and B. J. Gaffney, 1980, Pro. Natl. Acad. Sci. USA, 77:5060-5063, is characterized by a sharp drop in the second moment at 12 degrees C as a result of increasing the temperature. Interesting features of this phase transition are a hysteresis of 11 K and extremely slow kinetics. It is interpreted as the onset of a flip-flop of the hydrocarbon chains about their long axis. At the pretransition, this type of motion is assumed to change into a fast rotation. The proposed models for the three phases are confirmed by computer calculations of theoretical values for the second and fourth moments of the corresponding NMR signals.     

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.     

Deuterium magnetic resonance study of the gel and liquid crystalline phases of dipalmitoyl phosphatidylcholine.

Deuterium magnetic resonance is applied to the study of the liquid crystalline and gel phases, and of the phase transition, of a multilamellar dispersion of chain perdeuterated (d62)-dipalmitoyl phosphatidylcholine/H2O. Analysis of the deuterium spectra in terms of the moments of the spectra allows one to make quantitative statements concerning the distribution of quadrupolar splittings even in complicated situations, e.g., when using perdeuterated sampled or when there are mixed phases. This analysis indicates that d62-dipalmitoyl phosphatidylcholine in excess H2O undergoes a sharp phase transition (with a width of less than 1 degree C) at approximately 37 degrees C and that there appears to be hysteresis in the phase transition of approximately 1 degree C. In the lamellar liquid crystalline phase above 37 degrees C the spectra show a number of well-resolved features whose quadrupolar splittings can be followed as the temperature is varied. The gel phase near 20 degrees C possesses a very broad, almost featureless spectrum that does not seem to support a model of the gel phase wherein the hydrocarbon chains are fully extended in the all-trans conformation. At temperatures near 0 degrees C the spectra clearly indicate that a large fraction of the lipid molecules cease the rotation about their long axes, giving a spectrum more characteristic of a rigid or solid sample. These results give a picture of the gel phase as a phase characterized by considerable hydrocarbon chain disorder near 20 degrees C and becoming a more solid-like phase near 0 degrees C. The spin-lattice relaxation time, T1, has been measured at 20 degrees C in the gel phase, and at 37 and 45 degrees C in the liquid crystalline phase. The values of T1 obtained for each of the resolvable peaks in the spectrum at 37 degrees C are compared to the values (for each peak) of T2e, the decay time of the quadrupolar echo, obtained at the same temperature. These results are discussed in terms of a simple two-motion model.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles.

6-Carboxyfluorescein was employed to examine the effect of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles. Proton permeability was measured by monitoring the decrease of 6-carboxyfluorescein fluorescence after a pH gradient from 3.5 (outside the vesicle) to 8.0 (inside the vesicle) was established. At 20 degrees C and below 1.2 M ethanol, the fluorescence decrease is best described by a single exponential function. Above 1.2 M ethanol, the intensity decrease is better described by a two-exponential decay law. Using the fitted rate constants and the vesicle radii determined from light-scattering measurements, the proton permeability coefficient, P, in DPPC vesicles was calculated as a function of ethanol concentration. At 20 degrees C, P increases monotonically with increasing ethanol content up to 1.0 M, followed by an abrupt increase at 1.2 M. The vesicle size also exhibits a sudden increase at around 1.2 M ethanol, which has been shown to result from vesicle aggregation rather than vesicle fusion. The abrupt increases in P and in vesicle size occur at the concentration region close to the critical ethanol concentration for the formation of the fully interdigitated gel state of DPPC. At 14 degrees C, the abrupt change in P shifts to 1.9-2.0 M ethanol, completely in accordance with the ethanol-temperature phase diagram of interdigitated DPPC. Effects of methanol and benzyl alcohol on lipid interdigitation have also been examined. At 20 degrees C, DPPC large unilamellar vesicles exhibit a dramatic change in P at 3 M methanol and at 40 mM benzyl alcohol. These concentrations come close to the critical methanol and benzyl alcohol concentrations for the formation of fully interdigitated DPPC structures determined previously by others. It can be concluded that proton permeability increases dramatically as DPPC is transformed from the noninterdigitated gel to the fully interdigitated gel state by high concentrations of alcohol. This marked increase in proton permeability can be attributed to the combined effect of the changes in membrane thickness and surface charge density, due to the ethanol-induced lipid interdigitation. The possible effects of the increased proton permeability caused by ingested ethanol on gastric mucosal membranes are discussed.     

The interfacial structure of phospholipid bilayers: differential scanning calorimetry and Fourier transform infrared spectroscopic studies of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine and its dialkyl and acyl-alkyl analogs.

The thermotropic phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) and its 1,2-dialkyl, 1-acyl 2-alkyl and 1-alkyl 2-acyl analogs was examined by differential scanning calorimetry, and the organization of these molecules in those hydrated bilayers was studied by Fourier transform infrared spectroscopy. The calorimetric data indicate that substitution of either or both of the acyl chains of DPPC with the corresponding ether-linked hydrocarbon chain results in relatively small increases in the temperature (< 4 degrees C) and enthalpy (< 1 kcal/mol) of the lipid chain-melting phase transition. The spectroscopic data reveal that replacement of one or both of the ester-linked hydrocarbon chains of DPPC with its ether-linked analog causes structural changes in the bilayer assembly, which result in an increase in the polarity of the local environments of the phosphate headgroups and of the ester carbonyl groups at the bilayer polar/apolar interface. The latter observation is unexpected, given that ester linkages are considered to be intrinsically more polar that ether linkages. This finding cannot be satisfactorily rationalized unless the conformation of the glycerol backbones of the analogs containing ether-linked hydrocarbon chains differs significantly from that of diacyl glycerolipids such as DPPC. A comparison of the alpha-methylene scissoring bands and the methylene wagging band progressions of these lipids with the corresponding absorption bands of specifically chain-perdeuterated analogs of DPPC also supports the conclusion that replacement of the ester-linked hydrocarbon chains of DPPC with the corresponding ether-linked analog induces conformational changes in the lipid glycerol backbone. The suggestion that the conformation of glycerol backbones in the alkyl-acyl and dialkyl derivatives of DPPC differs from that of the naturally occurring 1,2-diacyl glycerolipid suggests that mono- and di-alkyl glycerolipids may not be good models of their diacyl analogs. These results, and previously published evidence that DPPC analogs with ether-linked hydrocarbon chains spontaneously form chain-interdigitated gel phases at low temperatures, clearly indicate that the properties of lipid bilayers can be substantially altered by small changes in the chemical structures of their polar/polar interfaces, and highlight the critical role of the interfacial region as a determinant of the structure and organization of lipid assemblies.     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I' band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 degrees C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 degrees C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 degrees C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

Thermotropic behavior of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles exerts a profound effect upon the DPPC gel to liquid-crystalline phase transition. The glycoprotein was reconstituted into DPPC vesicles by octyl glucoside dialysis. The gel to liquid-crystalline phase transition of these vesicles was monitored by differential scanning calorimetry. Vesicles formed in the absence of glycoprotein (600--2100-A diameter) underwent the phase transition at 41.0 degrees C and had an associated enthalpy change of 8.0 +/- 1.6 kcal/mol. Increasing the mole ratio of glycoprotein to DPPC in the vesicles to 0.15 mol % reduced both the transition temperature and the transition enthalpy change. The enthalpy change as a function of the mole percent glycoprotein could be fit to a straight line by a least-squares procedure. Extrapolation of the results to the glycoprotein concentration where the enthalpy change was zero indicated one glycoprotein molecule bound 270 +/- 150 molecules of DPPC.     

Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)