Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectroscopy of selectively deuterated dimyristoylphosphatidylcholine: studies on dimyristoylphosphatidylcholine-cholesterol bilayers

Dimyristoylphosphatidylcholine (DMPC), selectively deuterated in the sn-2 chain at the 3, 6, and 10 positions is used to probe DMPC-cholesterol interactions in multilamellar dispersions. Using the Raman spectral linewidths of the 2100 cm-1 C2H2 stretching modes as an index of membrane disorder, we demonstrate that cholesterol tends to order, or increase the number of trans carbon-carbon bonds within the DMPC acyl chain near the headgroup region at all temperatures. At low temperatures, cholesterol disorders the acyl chains near the methyl termini by inducing gauche conformers; cholesterol orders the entire chain at higher temperatures. These determinations are qualitatively consistent with conclusions drawn from deuterium nuclear magnetic resonance studies, but specifically reflect acyl chain trans/gauche isomerization on the 10(-12)-10(-13) s vibrational time scale.     

Quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by infrared spectroscopy

A method is proposed and demonstrated for the direct determination of conformational disorder (trans-gauche isomerization) as a function of acyl-chain position in phospholipid bilayer membranes. Three specifically deuterated derivatives of dipalmitoylphosphatidylcholine (DPPC), namely 4,4,4',4'-d4-DPPC (4-d4-DPPC), 6,6,6',6'-d4-DPPC (6-d4-DPPC), and 10,10,10',10'-d4-DPPC (10-d4-DPPC), have been synthesized. The CD2 rocking modes in the Fourier transform infrared (FT-IR) spectrum have been monitored as a function of temperature for each derivative. A method originally applied by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of hydrocarbon chain conformation in alkanes has been used to analyze the data. The rocking modes appear at 622 cm-1 for a CD2 segment surrounded by a trans C-C-C skeleton and between 645 and 655 cm-1 for segments surrounded by particular gauche conformers. The integrated band intensities of these modes have been used to monitor trans-gauche isomerization in the acyl chains at particular depths in the bilayer. At 48 degrees C, above the gel-liquid-crystal phase transition, the percentage of gauche rotamers present is 20.7 +/- 4.2, 32.3 +/- 2.3, and 19.7 +/- 0.8 for 4-d4-DPPC, 6-d4-DPPC, and 10-d4-DPPC, respectively. The gel phase of the latter two molecules is highly ordered. In contrast, a substantial population of gauche rotamers was observed for the 4-d4-DPPC. The conformational analysis yields a range of 3.6-4.2 gauche rotamers/acyl chain of DPPC above the phase transition. This range is in excellent accord with the dilatometric data of Nagle and Wilkinson [(1978) Biophys. J. 23, 159-175]. The significant advantages of the FT-IR approach are discussed.     

Acyl chain conformational ordering in 1,2 dipalmitoylphosphatidylethanolamine. Integration of FT-IR and 2H NMR results.

The extent of trans-gauche isomerization at the 4 and 4' positions of the acyl chains of fully hydrated 4,4,4',4'-d4 1,2-dipalmitoylphosphatidylethanolamine (4-d4 DPPE) bilayers was quantitatively evaluated from the infrared (IR) intensity of the CD2 rocking modes. About 20% gauche conformers were observed at 72 degrees C (above Tm), while at 23 degrees C, well below Tm, about 4% were noted. The order parameter SC-D was determined from 2H nuclear magnetic resonance (NMR) quadrupolar splittings. SC-D is the product of a segmental order parameter (S gamma), which depends on conformational order, and a chain order parameter (S alpha) which depends on slower motions such as chain wobble. The IR-determined percentage of gauche forms was converted into a segmental order parameter and factored out of the measured value for SC-D to yield an estimate of S alpha = 0.59 for L alpha phase DPPE. A comparison with S alpha for 1,2-dipalmitoylphosphatidylcholine (DPPC) suggests that increased wobble is responsible for enhanced motional averaging of the quadrupolar splittings in the latter at a similar reduced temperature. The extent of conformational disordering [at the 4(4') position] is essentially unchanged between the two molecules. The current study demonstrates the advantage of integrating quantitative IR with 2H NMR data, for elucidation of the contributions of the individual motions that average the NMR quadrupolar splittings.     

A quantitative infrared determination of acyl chain conformation in gramicidin/dipalmitoylphosphatidylcholine mixtures.

A quantitative infrared characterization of phospholipid acyl chain disordering in 6,6,6'6'-d4 dipalmitoylphosphatidylcholine/ Gramicidin D bilayers has been made. Three CD2 rocking modes, at 622 cm-1, 646-649 cm-1, and 651-653 cm-1, assigned to particular conformers, were used to determine disorder in the presence of peptide, as well as percentages of particular classes of conformer within the total gauche population. At 44C, the gauche percentages in 10:1 and 30:1 lipid/peptide mixtures were 15% and 17%, respectively. At 34C, the corresponding values were 9.8% and 2.6%. The percentage of (single gauche bend + kink) conformers, relative to multiple gauche forms, decreases dramatically from 78% in the 30:1 mixture to 15% in the 10:1 mixture at 44C. These data provide the first quantitative measure of the extent to which a membrane-spanning peptide disorders phospholipid gel phases and orders liquid crystal phases.     

Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate

Using the molecular dynamics simulation technique, we studied the changes occurring in a dipalmitoylphosphatidylcholine (DPPC):cholesterol (CH) membrane at 50 mol% sterol when cholesterol is replaced with cholesterol sulfate (CS). Our simulations were performed at constant pressure and temperature on a nanosecond time scale. We found that 1) the area per DPPC:CS heterodimer is greater than the area of the DPPC:CH heterodimer; 2) CS increases ordering of DPPC acyl chains, but to a lesser extent than CH; 3) the number of hydrogen bonds between DPPC and water is decreased in a CS-containing membrane, but CS forms more water hydrogen bonds than CH; and 4) the membrane dipole potential reverses its sign for a DPPC-CS membrane compared to a DPPC-CH bilayer. We also studied the changes occurring in lipid headgroup conformations and determined the location of CS molecules in the membrane. Our results are in good agreement with the data available from experiments.     

The influence of oxygenated sterol compounds on dipalmitoylphosphatidylcholine bilayer structure and packing

Fourier Transform Infra-red and Raman Spectroscopies indicate that 7 alpha-hydroxycholesterol and 7-ketocholesterol have a diminished capacity to condense (increase the packing order of) fluid-state dipalmitoylphosphatidylcholine (DPPC) acyl chains when compared with the effects of cholesterol and the other oxidized sterols studied. DPPC head groups were also more ordered by 7-ketocholesterol over the temperature range 10 degrees - 70 degrees C. Primary effects of these sterols appear to be associated with the hydrophillic regions of the DPPC bilayer, although packing arrangements with acyl chains are also involved. Phosphate and acyl chain ester groups were observed to possess a packing order which was invariant which indicates that these may be the target groups in the interaction with 7-ketocholesterol. A surprising observation was the synergistic amplification of the effects of 7-ketocholesterol by the presence of cholesterol in the DPPC bilayer.     

Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine

The stoichiometric palmitoyllysophosphatidylcholine (lysoPC)/gramicidin (4:1, mol/mol) lamellar complex (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141-144) is a useful model system to investigate the various aspects of lipid protein interactions. To study the effect of gramicidin on local order and motion of 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) we employed 31P and 2H nuclear magnetic resonance (NMR) using selectively deuterated lysoPC's and we compared the results to those obtained for lysoPC in bilayers with cholesterol (1:1, mol/mol) and dipalmitoylphosphatidylcholine (DPPC) (1:4, mol/mol). 2H-NMR experiments on acyl chain deuterated lysoPC showed similar quadrupole splittings in the liquid crystalline state for the lysoPC/DPPC and the lysoPC/gramicidin samples. In the lysoPC/cholesterol sample an increase of the quadrupole splitting was found. T1 measurements showed that gramicidin decreases the lysoPC acyl chain motion, especially at the C12 position. In the lysoPC/cholesterol sample an increase of motion was observed as compared to lysoPC in fluid bilayers of DPPC. 31P-NMR and 2-H-NMR measurements of lysoPC, deuterated at the alpha- and beta-position of the choline moiety, indicated an increase in headgroup flexibility in all samples as compared to the parent compound DPPC. In addition, a change in headgroup conformation was observed. The alpha- and beta-segments in all samples exhibited concerted motion. It was found that also in the polar headgroup gramicidin induces a decrease of the rate of motion.     

Structure of dipalmitoylphosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation.

By using molecular dynamics simulation technique we studied the changes occurring in membranes constructed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol at 8:1 and 1:1 ratios. We tested two different initial arrangements of cholesterol molecules for a 1:1 ratio. The main difference between two initial structures is the average number of nearest-neighbor DPPC molecules around the cholesterol molecule. Our simulations were performed at constant temperature (T = 50 degrees C) and pressure (P = 0 atm). Durations of the runs were 2 ns. The structure of the DPPC/cholesterol membrane was characterized by calculating the order parameter profiles for the hydrocarbon chains, atom distributions, average number of gauche defects, and membrane dipole potentials. We found that adding cholesterol to membranes results in a condensing effect: the average area of membrane becomes smaller, hydrocarbon chains of DPPC have higher order, and the probability of gauche defects in DPPC tails is lower. Our results are in agreement with the data available from experiments.     

Nuclear magnetic resonance and calorimetric study of the structure, dynamics, and phase behavior of uranyl ion/dipalmitoylphosphatidylcholine complexes.

The interaction of UO2(2+) with dipalmitoylphosphatidylcholine (DPPC) has been studied as a function of temperature and composition using nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), and monolayer studies. Computer simulations of the 31P-NMR powder spectra of DPPC dispersions in the presence of various concentrations of UO2(2+) are consistent with the binding stoichiometry of [UO2(2+)]/[DPPC] = 1:4 at [UO2(2+)]/[DPPC] less than 0.3. This complex undergoes a phase transition to the liquid crystalline phase at T'm = 50 +/- 3 degrees C with a breadth delta T'm = 7 +/- 3 degrees C. This broad transition gradually disappears at higher UO2(2+) concentrations, suggesting the presence of yet another UO2(2+)/DPPC complex (or complexes) whose NMR spectra are indistinguishable from those of the 1:4 UO2(2+)/DPPC species. The temperature-dependent 13C powder spectra of 2(1-13C) DPPC dispersions in the presence of 1.2 mol ratio of UO2(2+) show that this higher order complex (complexes) also undergoes a phase transition to the liquid crystalline state at T'm +/- = 58 +/- 3 degrees C with a breadth delta T"m = 15 +/- 5 degrees C. The NMR spectra indicate that exchange among these various UO2(2+)/DPPC complexes is slow. In addition, computer simulations of the 31P-, 13C-, and 2H-NMR powder spectra show that axial diffusion of the DPPC molecules about their long axes is quenched by addition of UO2(2+) and acyl chain isomerization is the dominant motional mode. The isomerization is best described as two-site hopping of the greater than C-D bond at a rate of approximately 10(6) s-1, a motional mode which is expected for a kink diffusion.     

Molecular packing in steroid-lecithin monolayers,part II: Mixed films of cholesterol with dipalmitoylphosphatidylcholine and tetradecanoic acid

Mixed film studies of the systems cholesterol/tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine have been carried out over the entire compositional range at 21°C. When compared on an acyl chain basis the condensing effects were found to be essentially independent of which host-lipid was utilized. The phase change of the host lipid was shifted to higher pressures, then broadened and eliminated. Maximal condensation occurred at just above 42 mol% for the cholesterol/DPPC system. In both systems the two components were initially found to be miscible at all proportions.The results are interpreted in terms of the molecular packing of cholesterol with acyl boundary layers, one significantly, one weakly affected. Maximum condensation is a result of packing that provides maximum cholesterol/acyl chain contact. Consideration is given to both long term stability of such mixed monolayers and the behaviour of the corresponding bilayer states.     

A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of cholesterol (Chol) and ergosterol (Erg) on the thermotropic phase behavior and organization of DPPC bilayers. Ergosterol is the major sterol in the biological membranes of yeasts, fungi and many protozoa. It differs from Chol in having two additional double bonds, one in the steroid nucleus at C7-8 and another in the alkyl chain at C22-23. Erg also has an additional methyl group in the alkyl chain at C24. Our DSC studies indicate that the incorporation of Erg is more effective than Chol is in reducing the enthalpy of the pretransition. At lower concentrations Erg is also more effective than Chol in reducing the enthalpies of both the sharp and broad components of main phase transition. However, at sterol concentrations from 30 to 50 mol%, Erg is generally less effective at reducing the enthalpy of the broad components and does not completely abolish the cooperative hydrocarbon chain-melting phase transition at 50 mol%, as does Chol. Nevertheless, in this higher ergosterol concentration range, there is no evidence of the formation of ergosterol crystallites. Our FTIR spectroscopic studies demonstrate that Erg incorporation produces a similar ordering of liquid-crystalline DPPC bilayers as does Chol, but an increased degree of hydrogen bonding of the fatty acyl carbonyl groups in the glycerol backbone region of the DPPC bilayer. These and other results indicate that Erg is less miscible in DPPC bilayers at higher concentrations than is Chol. Finally, we provide a tentative molecular explanation for the comparative experimental and computation results obtained for Erg and Chol in phospholipid bilayers, emphasizing the dynamic conformational differences between these two sterols.     

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.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Calcium ion interactions with insoluble phospholipid monolayer films at the A/W interface. External reflection-absorption IR studies.

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.     

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.     

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The effect of lung surfactant apolipoproteins B/C on the chemical shift anisotropy of sn-2-[1-13C]dipalmitoylphosphatidylcholine

Nuclear magnetic resonance spectroscopy has been used to investigate the effect of the lung surfactant apolipoproteins B/C on dipalmitoylphosphatidylcholine to address the mechanism by which the adsorption rate of phospholipids from the bulk to the air/water interface is enhanced. Apolipoproteins B/C were isolated from bovine lung and separated from associated lipids by lipophilic Sephadex column chromatography. Amino acid analysis indicated the presence of both apolipoproteins B and C. The 13C chemical shift anisotropy of DPPC was determined as a function of temperature. Previous workers (Wittebort et al., Biochemistry, 20 (1981) 3487-3502) have concluded that the observed magnitude of the chemical shift anisotropy of the carbonyl group of the sn-2 acyl chain in pure DPPC is a result of rapid rotation about an axis along the length of the phospholipid both in the gel and liquid crystalline state. The orientation of the carbonyl group with respect to the axis of diffusion, however, undergoes an approximately 25-30 degrees shift in passage from the gel to liquid crystalline state, with the intermediate, rippled (P beta') state composed of an exchange between these two orientations. The presence of physiological concentrations SP-B/C reduced the width of the anisotropy of DPPC below but had no effect on lipids above the main phase transition temperature. This suggests that SP-B/C has a general effect on the entire assembly of lipids. The temperature of the onset of the orientational change is lowered indicating a portion of the lipids are affected by the lung surfactant apolipoproteins.     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)