Unusual partitioning behavior of CaATPase in dipalmitoylphosphatidylethanolamine/dielaidoylphosphatidylcholine++ + mixtures.

CaATPase from rabbit sarcoplasmic reticulum has been isolated, purified, stripped of its native lipids, and reconstituted into binary lipid mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoylphosphatidylethanolamine (DPPE) or acyl-chain perdeuterated DPPE (DPPE-d62). The partitioning properties of the protein were determined from differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. Acyl-chain perdeuteration allows the separate determination of the order and melting characteristics of each lipid species with FT-IR. The binary lipid mixture has been shown to be phase separated in the gel state (Brauner, J. W., and R. Mendelsohn, 1986, Biochim. Biophys. Acta, 861:16-24). The solid phases present at low temperatures correspond to a pure DEPC phase and a mixed phase of DEPC/DPPE-d62. Insertion of protein at 37 degrees C leads to a domain of relatively protein-free DPPE-d62 and a phase containing both lipids plus CaATPase. We suggest that CaATPase selects a fixed composition (60% DEPC, 40% DPPE-d62) for its immediate environment. The composition of the lipid in the immediate vicinity of protein is largely independent of the initial DEPC/DPPE-d62 ratios in the reconstitution protocol. The relevance of these results to observations of discrete domains in native membranes is discussed.     

Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

Fourier transform infrared spectroscopic identification of gel phase domains in reconstituted phospholipid vesicles containing Ca2+-ATPase

Ca2+-ATPase from rabbit sarcoplasmic reticulum has been isolated, purified, and reconstituted into lipid environments containing as primary components 1,2-dielaidoylphosphatidylcholine (DEPC) and acyl-chain perdeuterated 1,2-dimyristoylphosphatidylcholine (DMPC-d54). Differential scanning calorimetry (DSC) has been used to elucidate the phase behavior of this lipid pair while Fourier transform infrared spectroscopy (FT-IR) has been used to monitor the state of each lipid component in the presence of protein. The lipid mixture shows gel state miscibility over at least most of the composition range, a result in good accord with Van Dijck et al. (Biochim. Biophys. Acta 470, 58-69 (1977)), for the binary mixture with proteated DMPC. Acyl chain perdeuteration thus does not greatly alter the miscibility properties of the lipid pair. Reconstitution of Ca2+-ATPase with this lipid pair proceeds with moderate efficiency. Up to 80% of the endogenous lipid can be replaced depending on the lipid composition. Unusual composition-dependent protein-induced effects on lipid melting properties are noticed. At low levels of DMPC-d54, both the DEPC and DMPC-d54 components have their melting processes broadened and shifted to lower temperatures, compared with binary lipid mixtures of the same composition. This suggests that protein perturbs both lipids in similar fashion. At high levels of DMPC-d54, the DEPC component exhibits a highly cooperative melting process at temperatures close to that for pure DEPC. This strongly indicates that domains of DEPC are present (at least at low temperatures) in the bilayer, and that Ca2+-ATPase is excluded from these domains. The protein thus exhibits preferential interaction with the DMPC-d54 component. This work demonstrates the utility of FT-IR for identification of the molecular origin of particular domains in reasonably complex lipid mixtures. The relevance of this work to native membrane systems where lipid domains have been observed by several groups is discussed.     

Fourier-transform infrared studies of CaATPase partitioning in phospholipid mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 with 1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-stearoyl-2-oleoylphosphatidylcholine

CaATPase from rabbit sarcoplasmic reticulum has been reconstituted into binary lipid mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1,2-dipalmitoylphosphatidylcholine-d62 (DPPC-d62) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC)/DPPC-d62. Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor temperature-induced structural alterations in the individual lipid components in the presence and absence of protein. A simple two-state model is used to construct a phase diagram that is in good agreement with one constructed from differential scanning calorimetry data, for the POPE/DPPC-d62 (protein-free) system. Although these two lipids are miscible over at least most of the composition range, substantial deviations from ideal behavior are observed. An estimate of the nonideality of mixing in both the gel and liquid-crystalline phases is obtained from regular solution theory. The phase diagram for SOPC/DPPC-d62 shows gel-phase immiscibility. FT-IR studies of ternary (POPE/DPPC-d62/CaATPase) complexes indicate that both lipid components are disordered by protein at all temperatures studied. In addition, their melting events are broadened and shifted to lower temperatures compared with the appropriate binary lipid mixture. Semiquantitative estimates for the fraction of each lipid melted are obtained from the model. The effect of protein on SOPC/DPPC-d62 mixtures depends on that total lipid to protein ratio. At low protein levels, SOPC is preferentially selected by CaATPase, so that bulk lipid is enriched in DPPC-d62. At high levels of protein, both lipid components are selected. The applicability of vibrational spectroscopy for determination of the partitioning preferences of membrane proteins into regions of particular chemical structure or physical order in a complex lipid environment is demonstrated.     

Fourier transform infrared spectroscopic identification of gel phase domains in reconstituted phosphilipid vesicles containing Ca2+-ATPase

Ca²⁺-ATPase from rabbit sarcoplasmic reticulum has been isolated, purified, and reconstituted into lipid environments containing as primary components 1,2-dielaidoylphosphatidylcholine (DEPC) and acyl-chain perdeuterated 1,2-dimyristoylphosphatidylcholine (DMPC-d₅₄). Differential scanning calorimetry (DSC) has been used to elucidate the phase behavior of this lipid pair while Fourier transform infrared spectroscopy (FT-IR) has been used to monitor the state of each lipid component in the presence of protein. The lipid mixture shows gel state miscibility over at least most of the composition range, a result in good accord with Van Dijck et al. (Biochim. Biophys. Acta 470, 58–69 (1977)), for the binary mixture with proteated DMPC. Acyl chain perdeuteration thus does not greatly alter the miscibility properties of the lipid pair. Reconstitution of Ca²⁺-ATPase with this lipid pair proceeds with moderate efficiency. Up to 80% of the endogenous lipid can be replaced depending on the lipid composition. Unusual composition-dependent protein-induced effects on lipid melting properties are noticed. At low levels of DMPC-d₅₄, both the DEPC and DMPC-d₅₄ components have their melting processes broadened and shifted to lower temperatures, compared with binary lipid mixtures of the same composition. This suggests that protein perturbs both lipids in similar fashion. At high levels of DMPC-d₅₄, the DEPC component exhibits a highly cooperative melting process at temperatures close to that for pure DEPC. This strongly indicates that domains of DEPC are present (at least at low temperatures) in the bilayer, and that Ca²⁺-ATPase is excluded from these domains. The protein thus exhibits preferential interaction with the DMPC-d₅₄ component. This work demonstrates the utility of FT-IR for identification of the molecular origin of particular domains in reasonably complex lipid mixtures. The relevance of this work to native membrane systems where lipid domains have been observed by several groups is discussed.     

Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant

The thermotropic behavior of intact bovine lung surfactant and its hydrophobic extract has been monitored via the temperature dependence of the 2850 cm-1 phospholipid acyl chain CH2 symmetric stretching frequencies in the IR spectrum. A broad, reversible, melting event was noted from about 15 to 40 degrees C in both the lipid extract and the native surfactant. Slight protein-induced disordering of the lipid acyl chains was evident. The melting event was confirmed by differential scanning calorimetry. The major surfactant protein, a 30-36-kDa class of glycoprotein (SP-A), has been isolated from bovine lung lavage and purified by affinity chromatography. SP-A was reconstituted into a binary lipid mixture of acyl chain perdeuterated dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC-d62/DPPG, 85:15 w/w), a ratio which approximates that in surfactant. Use of DPPC-d62 permitted the FT-IR determination of the effect of protein on the thermotropic behavior of individual phospholipids in the binary mixture. High levels of SP-A induced an ordering of the phospholipids, as shown by an increase in the transition temperature of DPPC-d62 compared to the lipid model. In contrast, a mixture of the other surfactant proteins induced a progressive disordering of the phospholipids and disruption of the cooperativity of the melting event. Transition widths of about 3 degrees, 9 degrees, and 27 degrees were noted for protein:lipid ratios of 0, 1:1, and 2:1 (w/w), respectively. Possible roles for the various proteins in surfactant function are discussed in light of these data.     

Comparison of DPPC and DPPG environments in pulmonary surfactant models

Deuterium nuclear magnetic resonance was used to monitor lipid acyl-chain orientational order in suspensions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) containing Ca(2+) and the lung surfactant proteins SP-A and SP-B separately and together. To distinguish between protein-lipid interactions involving the PC and PG lipid headgroups and to examine whether such interactions might influence spatial distribution of lipids within the bilayer, acyl chains on either the DPPC or the DPPG component of the mixture were deuterated. The lipid components of the resulting mixtures were thus either DPPC-d(62)/DPPG (7:3) or DPPC/DPPG-d(62) (7:3), respectively. SP-A had little effect on DPPC-d(62) chain order but did narrow the temperature range over which DPPG-d(62) ordered at the liquid-crystal-to-gel transition. No segregation of lipid components was seen for temperatures above or below the transition. Near the transition, though, there was evidence that SP-A promoted preferential depletion of DPPG from liquid crystalline domains in the temperature range over which gel and liquid crystal domains coexist. SP-B lowered average chain order of both lipids both above and below the main transition. The perturbations of chain order by SP-A and SP-B together were smaller than by SP-B alone. This reduction in perturbation of the lipids by the additional presence of SP-A likely indicated a strong interaction between SP-A and SP-B. The competitive lipid-lipid, lipid-protein, and protein-protein interactions suggested by these observations presumably facilitate the reorganization of surfactant material inherent in the transformation from lamellar bodies to a functional surfactant layer.     

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: binary mixtures of dimyristoylphosphatidylethanolamine and tetramyristoylcardiolipin

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, anionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained zwitterionic phospholipid dimyristoylphosphatidylethanolamine (DMPE) were examined by a combination of differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After equilibration at low temperature, DSC thermograms exhibited by binary mixtures of TMCL and DMPE containing < 80 mol DMPE exhibit a fairly energetic lower temperature endotherm and a highly energetic higher temperature endotherm. As the relative amount of TMCL in the mixture decreases, the temperature, enthalpy and cooperativity of the lower temperature endotherm also decreases and is not calorimetrically detectable when the TMCL content falls below 20 mol%. In contrast, the temperature of the higher temperature endotherm increases as the proportion of TMCL decreases, but the enthalpy and cooperativity both decrease and the transition endotherms become multimodal. The FTIR spectroscopic results indicate that the lower temperature endotherm corresponds to a lamellar crystalline (L(c)) to lamellar gel (L(β)) phase transition and that the higher temperature transition involves the conversion of the L(β) phase to the lamellar liquid-crystalline (L(α)) phase. Moreover, the FTIR spectroscopic signatures observed at temperatures below the onset of the L(c)/L(β) phase transitions are consistent with the coexistence of structures akin to a TMCL-like L(c) phase and the L(β) phase, and with the relative amount of the TMCL-like L(c) phase increasing progressively as the TMCL content of the mixture increases. These latter observations suggest that the TMCL and DMPE components of these mixtures are poorly miscible at temperatures below the L(β)/L(α) phase transition temperature. Poor miscibility of these two components is also suggested by the complexity of the DSC thermograms observed at the L(β)/L(α) phase transitions of these mixtures and with the complex relationship between their L(β)/L(α) phase transition temperatures and the composition of the mixture. Overall, our data suggests that TMCL and DMPE may be intrinsically poorly miscible across a broad composition range, notwithstanding the homogeneity of the fatty acid chains of the two components and the modest (~10 °C) difference between their L(β)/L(α) phase transition temperatures.     

Molecular areas of phospholipids as determined by 2H NMR spectroscopy. Comparison of phosphatidylethanolamines and phosphatidylcholines.

The role of lipid diversity in biomembranes is one of the major unsolved problems in biochemistry. One parameter of possible importance is the mean cross-sectional area occupied per lipid molecule, which may be related to formation of nonbilayer structures and membrane protein function. We have used 2H NMR spectroscopy to compare the properties of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the L alpha phase. We find that DPPE has greater segmental order than DPPC, and that this increase in order is related to the smaller area per acyl chain found for DPPE. Values of the mean cross-sectional chain area are calculated using a simple diamond lattice model for the acyl chain configurational statistics, together with dilatometry data. The results obtained for the mean area per molecule are comparable with those from low angle x-ray diffraction studies.     

Pressure effects on dipalmitoylphosphatidylcholine bilayers measured by 2H nuclear magnetic resonance

The effects of pressure, up to 5 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-phosphatidylcholine perdeuterated in the acyl chains (DPPC-d62) were examined by using high-pressure NMR techniques. A deuterium probe was built, and the quadrupole splitting was measured against pressure at various temperatures. The experiments were performed on pure lipid bilayers in the liquid-crystalline state and on bilayers in the liquid-crystalline state containing the local anesthetic tetracaine. The results show that the order parameter of all segments of the acyl chains increases with pressure in the liquid-crystalline state. The more highly ordered regions of the chains are affected slightly more than the regions near the methyl ends. The addition of tetracaine increases the disorder of the chains, and pressure reverses the effect of anesthetic on the lipid as seen by the reversal of the changes in line shape and the measured order parameter.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

Differential scanning calorimetry and 2H NMR studies of the phase behavior of gramicidin-phosphatidylcholine mixtures

The extents of two-phase coexistence in the phase diagrams of mixtures of gramicidin with 1,2-bis(perdeuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-d62) and with 1,2-bis(perdeuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d54) mixtures have been explored with differential scanning calorimetry (DSC) and deuterium nuclear magnetic resonance (2H NMR). For both systems, increased gramicidin content causes a decrease in transition enthalpy and a broadening of the peak in excess heat capacity at the transition. In DMPC-d54-based mixtures, the broadening is roughly symmetric about the pure lipid transition temperature. Addition of gramicidin to DPPC-d62 extends the excess heat capacity peak on the low-temperature side, resulting in a slightly asymmetric scan. Deuterium NMR spectra showing a superposition of gel and liquid-crystalline components, observed for both mixtures, indicate the presence of two-phase coexistence. For the DPPC-d62-based mixtures, two-phase coexistence is restricted to an approximately 2 degrees C temperature range below the pure transition temperature. For DMPC-d54-based mixtures, the region of two-phase coexistence is even narrower. For both mixtures, beyond a gramicidin mole fraction of 2%, distinct gel and liquid-crystal contributions to the spectra cannot be distinguished. Along with the broad featureless nature of the DSC scan in this region, this is taken to indicate that the transition has been replaced by a continuous phase change. These results are consistent with the existence of a closed two-phase region having a critical concentration of gramicidin below 2 mol%.     

Thermotropic properties and molecular dynamics of cholesteryl ester rich very low density lipoproteins: effect of hydrophobic core on polar surface

Cholesteryl ester rich very low density lipoproteins (CER-VLDL), isolated from the plasma of rabbits fed a hypercholesterolemic diet, have been studied by differential scanning calorimetry (DSC), 13C nuclear magnetic resonance (NMR), and spin-label electron paramagnetic resonance (EPR) to determine the temperature-dependent dynamics of cholesteryl esters in the hydrophobic core and of phospholipids on the polar surface. Intact CER-VLDL exhibit two DSC heating endotherms; these occur at 40-42 and 45-48 degrees C. Cholesteryl esters isolated from CER-VLDL also exhibit two DSC endotherms; these occur at 50.0 and 55.1 degrees C and correspond to the smectic----cholesteric and cholesteric----isotropic liquid-crystalline phase transitions. A model mixture containing cholesteryl linoleate, oleate, and palmitate in a ratio (0.21, 0.51, and 0.28 mol fraction) similar to that in CER-VLDL exhibited comparable DSC endotherms at 45.2 and 51.5 degrees C. CER-VLDL at 37 degrees C gave 13C NMR spectra that contained no resonances assignable to cholesteryl ring carbons but detectable broad resonances for some fatty acyl chain carbons, suggesting the cholesteryl esters were in a liquid-crystalline state. When the mixture was heated to 42 degrees C, broad ring carbon resonances became detectable; at 48 degrees C, they became narrow, indicating the cholesteryl esters were in an isotropic, liquid-like state. With increasing temperature over the range 38-60 degrees C, the resonances for cholesteryl ring carbons C3 and C6 in CER-VLDL narrowed differentially. Similar spectral changes were observed for the synthetic cholesteryl ester mixture, except they occurred at temperatures about 10 degrees C higher. These results indicate that the two DSC transitions in CER-VLDL do not directly correlate with the smectic----cholesteric and cholesteric----isotropic transitions exhibited by pure cholesteryl esters. (5-Doxylpalmitoyl)-phosphatidylcholine (5-DP-PC) and (12-doxylstearoyl)phosphatidylcholine (12-DS-PC) were used to probe the polar surface monolayer of CER-VLDL; the corresponding cholesteryl esters (5-DP-CE and 12-DS-CE) were used to probe the hydrophobic core. None of these probes in CER-VLDL detected an abrupt change in EPR order parameters, S, or maximum splitting, 2T max, over the temperature range 20-58 degrees C even though 12-DS-PC and 5-DP-PC can detect phase transitions in phospholipid bilayers and 12-DS-CE and 5-DP-CE can detect phase transitions in neat cholesteryl esters. However, 12-DS-CE and 5-DP-CE did detect a much greater acyl chain order for the neutral lipids of CER-VLDL than for those of normal triglyceride-rich VLDL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.     

Raman spectroscopic studies of the packing properties of mixed dihexadecyl- and dipalmitoylphosphatidylcholine bilayer dispersions

X-ray diffraction studies suggest the existence of two separate gel phases for mixed dihexadecylphosphatidylcholine (DHPC)/dipalmitoylphosphatidylcholine (DPPC) bilayers [Kim, J. T., Mattai, J., & Shipley, G. G. (1987) Biochemistry 26, 6599-6603; Lohner, K., Schuster, A., Degovics, G., Müller, K., & Laggner, P. (1987) Chem. Phys. Lipids 44, 61-70]. In one gel phase the lipid chains are interdigitated, while the other gel phase exhibits the conventional bilayer form. We use Raman spectroscopy to provide a detailed molecular analysis of the intermolecular and intramolecular interactions of the DHPC and DPPC molecules within these mixed bilayers. Observation of the methylene chain C-H stretching modes of DHPC and the methylene chain C-D stretching modes of DPPC-d62 for various mixed DHPC/DPPC-d62 bilayers enables the packing characteristics and conformational order of each lipid to be monitored separately. The spectral data indicate that the packing properties of DPPC-d62 in the mixed-lipid bilayers remain relatively unchanged, while the intramolecular and intermolecular properties of DHPC change dramatically as a function of the composition of the DHPC/DPPC-d62 mixed bilayer. This is consistent with a model based upon the existence of three characteristic lipid types for the mixed-lipid system, namely, domains of pure DPPC-d62 and pure DHPC species with interface lipids or boundary regions between the bulk domains.     

Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) II. Peptide conformation by infrared spectroscopy

The conformation and orientation of synthetic monomeric human sequence SP-B(1-25) (mSP-B(1-25)) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B(1-25) in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B(1-25) had a major alpha-helical conformation plus regions of beta-sheet structure. These alpha-helical regions reoriented later during film compression than beta structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d(62):DOPG), the IR spectra of mSP-B(1-25) showed that a significant, concentration-dependent conformational change occurred when mSP-B(1-25) was incorporated into a DPPC-d(62):DOPG monolayer. At an mSP-B(1-25) concentration of 10 wt.%, the peptide assumed a predominantly beta-sheet conformation with no contribution from alpha-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B(1-25) to form alpha-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B(1-25), a substantial alpha-helical peptide structural component was observed, but regions of alpha and beta structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B(1-25), peptide conformation was predominantly alpha-helical and the helical regions reoriented later during compression than the remaining beta structural components. The increased alpha-helical structure of mSP-B(1-25) demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence.     

Liposome/DNA systems: correlation between association, hydrophobicity and cell viability

Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.     

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.     

Solid-liquid phase behavior of binary fatty acid mixtures 3. Mixtures of oleic acid with capric acid (decanoic acid) and caprylic acid (octanoic acid)

Solid-liquid phase behavior of binary mixtures of oleic acid (OA)/capric acid (C10A) and OA/caprylic acid (C8A) were investigated by means of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction. The phase diagram of OA/C10A mixture constructed from the DSC results suggested that a molecular compound with the composition of OA:C10A = 3:2 is formed in a solid phase, and OA and the molecular compound are miscible, while C10A and the molecular compound are completely immiscible. The formation of the molecular compound was supported by the IR spectroscopic observation, and a possible model of the structure was proposed on the basis of X-ray diffraction spectrum in small angle region. This compound formation is characteristic of the OA/C10A mixture, and may be attributed to the similarity of the acyl chain length of C10A to the lengths of Delta- and omega-chains of OA (i.e., the chain segments divided by cis-double bond). The mixture of OA and C8A, whose chain length is close to but shorter than the two chain segments of OA, provided a eutectic-type phase diagram showing a partial mixing of the two components in OA-rich region. Thermodynamic analysis of the liquidus line in the phase diagram exhibits a systematic trend for the non-ideality parameter of mixing with the variation of the chain length difference between OA and saturated fatty acid species.     

Miscibility and phase behavior of N-acylethanolamine/diacylphosphatidylethanolamine binary mixtures of matched acyl chainlengths (N=14, 16)

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and (31)P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by (31)P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with (31)P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65 degrees C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.