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.     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state 2H NMR

Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using (2)H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ(9)-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2' and 16' positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer. Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-(2)H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime ((2)H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type (2)H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.     

Amphipathic interactions of cannabinoids with membranes. A comparison between delta 8-THC and its O-methyl analog using differential scanning calorimetry, X-ray diffraction and solid state 2H-NMR.

The effects of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) and its biologically inactive O-methyl ether analog on model phospholipid membranes were studied using a combination of differential scanning calorimetry (DSC), small angle X-ray diffraction and solid state 2H-NMR. The focus of this work is on the amphipathic interactions of cannabinoids with membranes and the role of the free phenolic hydroxyl group which is the only structural difference between these two cannabinoids. Identically prepared aqueous multilamellar dispersions of phosphatidylcholines in the absence and presence of cannabinoids were used. The DSC thermograms and X-ray diffraction patterns of these preparations allowed us to detect the strikingly different manners in which these two cannabinoids affect the thermotropic properties and the thickness of the bilayer. In order study the effects of the cannabinoids on different regions of the bilayer, we used solid state 2H-NMR with four sets of model membranes from dipalmitoylphosphatidylcholine deuterated in different sites, viz., the choline trimethylammonium head group, or one of the following three groups in the acyl chains; the 2'-methylene, 7'-methylene, 16'-methyl groups. Analysis of quadrupolar splittings indicated that delta 8-THC resides near the bilayer interface and the inactive analog sinks deeper towards the hydrophobic region. The temperature dependence of the solid state 2H-NMR spectra showed that, during the bilayer phase transition, the disordering of the choline head groups is a separate event from the melting of the acyl chains, and that amphipathic interactions between delta 8-THC and the membrane separate these two events further apart in temperature. The inactive analog lacks the ability to induce such a perturbation.     

Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Fourier transform infrared study on the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-y) decanoyl]-sn-glycero-3-phosphatidylcholine

Fourier transform infrared (FTIR) spectroscopy was used to study the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphatidyl choline (PPDPC) in the temperature range of 3-30 degrees C. Several changes in the spectral features of PPDPC were observed. Major alterations analogous to the gel-to-liquid crystalline phase transition of saturated phosphatidylcholines were evident at approximately 16 degrees C in both the wavenumbers and the halfbandwidths of five different vibrational modes of PPDPC, viz. asymmetric and symmetric CH2 stretching, C = O stretching, and CH2 bending. Also the pyrene ring deformation mode changed at this temperature. Using Fourier self-deconvolution technique we resolved the carbonyl stretching mode into two bands at approx. 1741 and 1726 cm-1. These bands are due to conformational differences in the ester linkages of the two acyl chains, and are further assigned on the basis of literature data to the sn-1 and sn-2 carbonyl groups, respectively. The ratio of the relative intensities of these two bands is shown to depend on the phase state of the phospholipid.     

Effects of replacement of a double bond by a cyclopropane ring in phosphatidylethanolamines: a 2H NMR study of phase transitions and molecular organization

The thermotropic behavior and molecular properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine (PDSPE) have been investigated by 2H NMR spectroscopy using samples selectively labeled at the 5'-, 9'-, 10'-, and 16'-positions of the sn-2 chains. Comparison with the corresponding phosphocholine analogues (POPC and PDSPC), obtained as intermediate synthetic products, was used to monitor the role of the polar head group. Replacement of the choline moiety by ethanolamine increased the gel to liquid-crystal transition temperature by 10-32 degrees C and led to a significantly higher ordering of the fatty acyl chains in the liquid-crystalline bilayer state. The lateral compression effect, due to the smaller area per polar head group in PE, results in a bilayer to hexagonal phase transition at elevated temperatures. The effects on both PC and PE due to replacement of the olefinic group by a cyclopropane unit are similar. A decrease in the temperature of the gel to liquid-crystal phase transition, Tc, is observed upon introduction of a cyclopropane ring; it goes from 26 degrees C in POPE to approximately 10 degrees C in PDSPE. In addition, a very significant broadening of the transition profile is observed. These observations are consistent with the poor packing ability of mixed saturated and cyclopropane-containing chains due to the bulky substituent effect. The temperature of the bilayer-hexagonal phase transition of PE samples was decreased by 15-20 degrees C on replacement of oleoyl chains by dihydrosterculoyl chains at the sn-2 position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier transform infrared spectroscopy of 13C = O-labeled phospholipids hydrogen bonding to carbonyl groups

Fourier transform infrared spectroscopy has been used to characterize the carbonyl stretching vibration of DMPC, DMPE, DMPG, and DMPA, all labeled with 13C at the carbonyl group of the sn-2 chain. Due to the vibrational isotope effect, the 13C = O and the 12C = O vibrational bands are separated by ca. 40-43 cm-1. This frequency difference does not change when the labeling is reversed with the 13C = O group at the sn-1 chain. For lipids in organic solvents possible conformational differences between the sn-1 and sn-2 ester groups have no effect on the vibrational frequency of the C = O groups. In aqueous dispersion unlabeled phospholipids always show a superposition of two bands for the C = O vibration located at ca. 1740 and 1727 cm-1. These two bands have previously been assigned to the sn-1 and sn-2 C = O groups. FT-IR spectra of 13C-labeled phospholipids show that the vibrational bands of both, the sn-1 as well as the sn-2 C = O group, are clearly superpositions of at least two underlying components of different frequency and intensity. Band frequencies were determined by Fourier self-deconvolution and second-derivative spectroscopy. The difference between the component bands is ca. 11-17 cm-1. Again, the conformational effect as shown by reversed labeling is negligible with only 1-2 cm-1. The splitting of the C = O vibrational bands in H2O and D2O is caused by hydrogen bonding of water molecules to both C = O groups as shown by a comparison with spectra of model ester compounds in different solvents. To extract quantitative information about changes in hydration, band profiles were stimulated with Gaussian-Lorentzian functions. The chemical nature of the head group and its electronic charge have distinctive effects on the extent of hydration of the carbonyl groups. In the gel and liquid-crystalline phase of DMPC the sn-2 C = O group is more hydrated than the sn-1 C = O. This is accord with the conformation determined by X-ray analysis. In DMPG the sn-1 C = O group seems to be more accessible to water, indicating a different conformation of the glycerol backbone.     

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

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.     

The influence of acyl chain-length asymmetry on the phase transition parameters of phosphatidylcholine dispersions

The influence of acyl chain-length asymmetry on the thermodynamic parameters (Tm, delta H, and delta S) associated with the reversible main phase transition of aqueous dispersions prepared from saturated diacyl phosphatidylcholines was studied by high-resolution differential scanning calorimetry. Two series of saturated diacyl phosphatidylcholines, grouped according to their molecular weights of 678 and 706, with a total number of 25 molecular species were examined. The normalized acyl chain-length difference between the sn-1 and sn-2 acyl chains for a given phospholipid molecule in the gel-state bilayer is expressed quantitatively by the structural parameter delta C/CL, and the values of delta C/CL for the two series of lipids under study vary considerably from 0.04 to 0.67. When the value of delta C/CL is within the range of 0.09-0.40, it was shown that the thermodynamic parameters are, to a first approximation, a linear function of delta C/CL with a negative slope. In addition, the experimental Tm values and the predicted Tm values put forward by Huang (Biochemistry (1991) 30, 26-30) are in very good agreement. Beyond the point of delta C/CL = 0.41, the influence of acyl chain-length asymmetry on the thermodynamic parameters deviates significantly from a linear function. In fact, within the range of delta C/CL values of 0.42-0.67, the thermodynamic parameters in the Tm (or delta H) vs. delta C/CL plot were shown to be bell-shaped with the maximal Tm (or delta H) at delta C/CL = 0.57. These results are discussed in terms of changes in the acyl chain packing modes of various phosphatidylcholine molecules within the gel-state bilayer in excess water.     

The interfacial conformation and transbilayer movement of diacylglycerols in phospholipid bilayers

The interaction of diacylglycerols, primarily 1,2-dilauroyl-sn-glycerol (1,2-DLG), with egg phosphatidylcholine (PC) bilayers was studied by NMR spectroscopy and other physical techniques. In the low proportions used (less than or equal to 20 mol % with respect to total lipid), 1,2-DLG formed bilayers with PC with no hexagonal phase separation, as assessed by light, polarizing and electron microscopy, and 31P and 13C NMR spectroscopy. The 13C-carbonyl chemical shift of 90% [13C]carbonyl 1,2-DLG was monitored in small unilamellar vesicles as a function of relative DLG content (1.5-20%) and temperature (10-55 degrees C). The chemically inequivalent sn-1 and sn-2 carbonyls gave a single, narrow resonance in vesicles, in contrast to neat 1,2-DLG and 1,2-DLG in organic solvents, whose spectra showed two well-separated carbonyl resonances. The chemical shift of 1,2-DLG in PC shows that the carbonyl groups are proximal to the aqueous interface, necessitating orientation of the DLG molecule along the normal to the bilayer. Both carbonyl groups are H-bonded to H2O, but the secondary ester (sn-2) carbonyl is relatively more hydrated than the primary ester (sn-1) carbonyl. The 13C-carbonyl chemical shift data further suggest that the interfacial conformation resembles that of crystalline and liquid crystalline lamellar 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine and certain PCs, in which the glycerol backbone is perpendicular to the bilayer plane. This conformation is different from that of crystalline 1,2-dilauroyl-sn-glycerol, in which the glycerol backbone is parallel to the bilayer plane. Between 1.5 and 8% DLG in vesicles, the chemical shift of the 1,2-DLG carbonyl at a given temperature was constant. However, above 8% DLG the chemical shift at each temperature increased with increasing DLG concentration, suggesting increased hydration at higher DLG content. At low temperatures 13C NMR spectra of vesicles with the highest proportions of 1,2-DLG studied (15 and 20%) showed two DLG carbonyl resonances, which most likely represent 1,2-DLG on outer and inner leaflets of the vesicle bilayer. The two peaks collapsed into a single resonance by 38 degrees C, at which temperature the two environments equilibrate with a rate constant of approximately 60 s-1 (t1/2 approximately 10 ms). Thus, transbilayer movement of DLG is extremely fast compared with phospholipids. In vesicles the 1,3-isomer of DLG exhibited a narrow carbonyl peak slightly downfield from that of 1,2-DLG. Acyl chain migration from 1,2-DLG to 1,3-DLG was monitored directly in the vesicle by time-dependent NMR measurements.     

13C NMR studies of the thermal properties of a model high density lipoprotein. Apolipoprotein A-I-dimyristoylphosphatidylcholine complex

The most abundant lipid and protein components of human plasma high density lipoproteins are phosphatidylcholine and apolipoprotein A-I (A-I). Under appropriate conditions, A-I spontaneously associates with dimyristoylphosphatidylcholine (DMPC) to quantitatively form a lipid-protein complex with a DMPC/A-I molar ratio of 100:1. Differential scanning calorimetry of this complex reveals two broad thermal transitions centered at approximately 27 and 72 degrees C. 13C NMR spectra of the complex have been obtained above, at, and below the lower transition temperature. The 13C resonance arising from the 3' carbon of the fatty acyl chains is a doublet, split by approximately 0.2 ppm, suggesting that the 3' carbon nuclei occupy two magnetically inequivalent sites. By replacing the sn-2 fatty acyl chain with myristate selectively 13C-enriched at carbon 3', we have shown that the splitting is, in fact, a result of magnetic inequivalence of the two sites and have assigned the lower field resonance to the 3' carbon nucleus of the sn-2 chain. The temperature dependence of the NMR relaxation rates indicates that the endothermic transition at 27 degrees C is associated with increased motional freedom for the phospholipids within this complex. The temperature dependence of the fatty acyl chain methylene 13C chemical shifts suggests that the population of gauche conformers increases above the transition temperature. These dynamic and conformational changes are characteristic of gel----liquid crystalline phase transitions observed in pure phospholipid systems. For the DMPC-A-I complex at 37 degrees C, the chemical shifts of the fatty acyl C 4'- 11' methylene envelope and of the C 7' and C 13' resonances occur significantly downfield from the corresponding chemical shifts for the DMPC vesicle. These results suggest that the apoprotein rigidifies the acyl chains by increasing their number of trans conformers.     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0–70°C. In the low-temperature (L_c) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (L_c) phase to the intermediate-temperature (Lβ) phase at 25°C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L_β) to the high-temperature (L_α) phase at 37°C is a gel-to-liquid-crystalline phase transition analogous to the 41.5°C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the L_c phase. In the L_β and L_α phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C=O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A₂ on the basis of a preferred head group conformation.     

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.