An infrared spectroscopic study of the thermotropic phase behavior of phosphatidylcholines containing omega-cyclohexyl fatty acyl chains

The thermotropic phase behavior of an odd- and an even-numbered member of the homologous series of 1,2-di-omega-cyclohexylphosphatidylcholines was studied using Fourier transform infrared spectroscopy. The results obtained indicate that the pronounced discontinuities in the behavior of the odd- and even-numbered homologues observed by differential scanning calorimetry can be attributed to differences in the organization of their respective gel states. The single phase transition exhibited by the odd-numbered compounds upon heating is shown by infrared spectroscopy to be a direct transition from a condensed, subgel-like phase (Lc phase) to the liquid-crystalline state (L alpha phase). In contrast, the multiple transitions exhibited by the even-numbered homologues are shown to be due to the initial conversion of an L beta-like phase to a more loosely packed gel phase, followed by the acyl chain-melting transition. Moreover, the major changes in the interaction between the acyl chains, and in the organization of the interfacial region of the bilayers formed by the even-numbered homologue, occur at temperatures below that of the onset of the chain-melting phase transition. The infrared spectroscopic changes observed also suggest that above the chain-melting transition, the odd- and even-numbered homologues form similar liquid-crystalline phases that are more 'ordered' than those of normal saturated straight-chain phosphatidylcholines. Most likely this is because the large size and the intrinsic rigidity of the omega-cyclohexyl group reduces the conformational disorder of the liquid-crystalline state by 'dampening' all acyl chain motions. The formation of a relatively ordered liquid-crystalline state may be the critical property exploited by the thermoacidophylic organisms in which omega-cyclohexyl fatty acids naturally occur.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 1. Differential scanning calorimetric studies

The thermotropic phase behavior of aqueous dispersions of phosphatidylcholines containing one of a series of methyl iso-branched fatty acyl chains was studied by differential scanning calorimetry. These compounds exhibit a complex phase behavior on heating which includes two endothermic events, a gel/gel transition, involving a molecular packing rearrangement between two gel-state forms, and a gel/liquid-crystalline phase transition, involving the melting of the hydrocarbon chains. The gel to liquid-crystalline transition is a relatively fast, highly cooperative process which exhibits a lower transition temperature and enthalpy than do the chain-melting transitions of saturated straight-chain phosphatidylcholines of similar acyl chain length. In addition, the gel to liquid-crystalline phase transition temperature is relatively insensitive to the composition of the aqueous phase. In contrast, the gel/gel transition is a slow process of lower cooperativity than the gel/liquid-crystalline phase transition and is sensitive to the composition of the bulk aqueous phase. The gel/gel transitions of the methyl iso-branched phosphatidylcholines have very different thermodynamic properties and depend in a different way on hydrocarbon chain length than do either the "subtransitions" or the "pretransitions" observed with linear saturated phosphatidylcholines. The gel/gel and gel/liquid-crystalline transitions are apparently concomitant for the shorter chain iso-branched phosphatidylcholines but diverge on the temperature scale with increasing chain length, with a pronounced odd/even alternation of the characteristic temperatures of the gel/gel transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing cis-monounsaturated acyl chain homologues of oleic acid: differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of dioleoylphosphatidylcholine and six of its longer chain homologues was studied by differential scanning calorimetry and 31P nuclear magnetic resonance (NMR) spectroscopy. Aqueous dispersions of these compounds all exhibit a single endotherm upon heating but upon cooling exhibit at least two exotherms, both of which occur at temperatures lower than those of their heating endotherm. The single transition observed upon heating was shown by 31P NMR spectroscopy to be a net conversion from a condensed, subgel-like phase (Lc phase) to the liquid-crystalline state. Aqueous ethylene glycol dispersions of these compounds also exhibit single endotherms upon heating and cooling exotherms centered at temperatures lower than those of their corresponding heating endotherm. However, the behavior of the aqueous ethylene glycol dispersions differs with respect to their transition temperatures and enthalpies as well as the extent of "undercooling" observed, and there is some evidence of discontinuities in the cooling behavior of the odd- and even-numbered members of the homologous series. Like the aqueous dispersions, 31P NMR spectroscopy also shows that the calorimetric events observed in aqueous ethylene glycol involve net interconversions between an Lc-like phase and the liquid-crystalline state. However, the Lc phase formed in aqueous ethylene glycol dispersions exhibits a considerably broader powder pattern than that observed in water. This, together with the fact that the transition enthalpies of the aqueous ethylene glycol dispersions are considerably higher than those of the aqueous dispersions, indicates that these lipids form more ordered Lc phases in aqueous ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing omega-cyclohexyl fatty acids. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of 10 phosphatidylcholines containing omega-cyclohexyl-substituted acyl chains was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The presence of the omega-cyclohexyl group has a profound effect on the thermotropic phase behavior of these compounds in a manner dependent on whether the fatty acyl chains have odd- or even-numbered linear carbon segments. The thermotropic phase behavior of the odd-numbered phosphatidylcholines is characterized by a single heating endotherm that was shown to be a superposition of at least two structural events by calorimetric cooling experiments. 31P NMR spectroscopy also showed that the single endotherm of the odd-chain compounds is the structural equivalent of a concomitant gel-gel and gel to liquid-crystalline phase transition. The calorimetric behavior of the even-numbered phosphatidylcholines is characterized by a complex array of gel-state phenomena, in addition to the chain-melting transition, in both the heating and cooling modes. The gel states of these even-numbered compounds are characterized by a relatively greater mobility of the phosphate head group as seen by 31P NMR spectroscopy. The differences between the odd-numbered and even-numbered compounds are reflected in a pronounced odd-even alternation in the characteristic transition temperatures and enthalpies and in differences in their responses to changes in the composition of the bulk aqueous phase. Moreover, both the odd-numbered and even-numbered omega-cyclohexylphosphatidylcholines exhibit significantly lower chain-melting transition temperatures and enthalpies than do linear saturated phosphatidylcholines of comparable chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 1. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of nine dl-methyl branched anteisoacylphosphatidylcholines was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The calorimetric studies demonstrate that these compounds all exhibit a complex phase behavior, consisting of at least two minor, low-enthalpy, gel-state transitions which occur at temperatures just prior to the onset of the gel/liquid-crystalline phase transition. In addition, at still lower temperatures, anteisobranched phosphatidylcholines containing fatty acyl chains with an odd number of carbon atoms show a major, higher enthalpy, gel-state transition, which was assigned to a conversion from a condensed to a more loosely packed gel phase. No such transition was observed for the even-numbered compounds in aqueous dispersion, but when dispersed in aqueous ethylene glycol, a major gel-state transition is clearly discernible for two of the even-numbered phospholipids. The major gel-state transition exhibits heating and cooling hysteresis and is fairly sensitive to the composition of the bulk aqueous phase. 31P NMR spectroscopic studies indicate that the major gel-state transition is accompanied by a considerable change in the mobility of the phosphate head group and that, at temperatures just prior to the onset of the gel/liquid-crystalline phase transition, the mobility of the phosphate head group is comparable to that normally exhibited by the liquid-crystalline state of most other phospholipids. The temperatures at which the gel/liquid-crystalline phase transition occurs and the enthalpy change associated with this process are considerably lower than those of the saturated n-acyl-PC's of comparable acyl chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the thermotropic phase behavior of phosphatidylcholines containing 2-alkyl substituted fatty acyl chains: a new class of phosphatidylcholines forming inverted nonlamellar phases.

We have synthesized a number of 1,2-diacyl phosphatidylcholines with hydrophobic substituents adjacent to the carbonyl group of the fatty acyl chain and studied their thermotropic phase behavior by differential scanning calorimetry, 31P-nuclear magnetic resonance spectroscopy, and x-ray diffraction. Our results indicate that the hydrocarbon chain-melting phase transition temperatures of these lipids are lower than those of the n-saturated diacylphosphatidylcholines of similar chain length. In the gel phase, the 2-alkyl substituents on the fatty acyl chains seem to inhibit the formation of tightly packed, partially dehydrated, quasi-crystalline bilayers (Lc phases), although possibly promoting the formation of chain-interdigitated bilayers. In the liquid-crystalline state, however, these 2-alkyl substituents destabilize the lamellar phase with respect to one or more inverted nonlamellar structures. In general, increases in the length, bulk, or rigidity of the alkyl substituent result in an increased destabilization of the lamellar gel and liquid-crystalline phases and a greater tendency to form inverted nonlamellar phases, the nature of which depends upon the size of the 2-alkyl substituent. Unlike normal non-lamella-forming lipids such as the phosphatidylethanolamines, increases in the length of the main acyl chain stabilize the lamellar phases and reduce the tendency to form nonlamellar structures. Our results establish that with a judicious choice of a 2-alkyl substituent and hydrocarbon chain length, phosphatidylcholines (and probably most other so-called "bilayer-preferring" lipids) can be induced to form a range of inverted nonlamellar structures at relatively low temperatures. The ability to vary the lamellar/nonlamellar phase preference of such lipids should be useful in studies of bilayer/nonbilayer phase transitions and of the molecular organization of various nonlamellar phases. Moreover, because the nonlamellar phases can easily be induced at physiologically relevant temperatures and hydration levels while avoiding changes in polar headgroup composition, this new class of 2-alkyl-substituted phosphatidylcholines should prove valuable in studies of the physiological role of non-lamella-forming lipids in reconstituted lipid-protein model membranes.     

Thermotropic phase behavior of phosphatidylcholines with omega-tertiary-butyl fatty acyl chains.

The thermotropic phase behavior of a homologous series of phosphatidylcholines containing acyl chains with omega-tertiary butyl groups was studied by differential scanning calorimetry, Fourier transform infrared spectroscopy, and 31P-nuclear magnetic resonance spectroscopy (31P-NMR). Upon heating, aqueous dispersions of these lipids exhibit single transitions which have been identified as direct conversions from Lc-like gel phases to the liquid-crystalline state by both infrared and 31P-NMR spectroscopy. The calorimetric data indicate that the thermodynamic properties of the observed transition are strongly dependent upon whether the acyl chains contain an odd- or an even-number of carbon atoms. This property is manifest by a pronounced odd/even alternation in the transition temperatures and transition enthalpies of this homologous series of lipids, attributable to the fact that the odd-numbered compounds form gel phases that are more stable than those of their even-numbered counterparts. The spectroscopic data also suggest that unlike other lipids which exhibit the so-called odd/even effect, major odd/even discontinuities in the packing of the polymethylene chains are probably not the dominant factors responsible for the odd/even discontinuities exhibited by these lipids, because only subtle differences in the appropriate spectroscopic parameters were detected. Instead, the odd/even alternation in the physical properties of these lipids may be attributable to significant differences in the organization of the carbonyl ester interfacial regions of the lipid bilayer and to differences in the intermolecular interactions between the terminal t-butyl groups of the odd- and even-numbered homologues. Our results also suggest that the presence of the bulky t-butyl groups in the center of the lipid bilayer reduces the conformational disorder of the liquid-crystalline polymethylene chains, and promotes the formation of Lc-like gel phases. However, these Lc-like gel phases are considerably less ordered than those formed by saturated, straight-chain lipids.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 2. An infrared spectroscopy study

The thermotropic phase behavior of four members of the homologous series of dl-methyl anteisobranched phosphatidylcholines was investigated by Fourier transform infrared spectroscopy. The odd-numbered phosphatidylcholines exhibit spectral changes in two distinct temperature ranges, while their even-numbered counterparts exhibit spectral changes within only a single temperature range. The high-temperature transition observed in the odd-numbered phosphatidylcholines and the single thermotropic event characteristic of the phase behavior of their even-numbered counterparts are both identified as gel/liquid-crystalline phase transitions. The low-temperature event exhibited only by the odd-numbered phospholipids is identified as a gel/gel phase transition that involves changes in the packing mode of the acyl chain methylene groups, as well as changes in the conformation of the glycerol ester interface. These infrared spectroscopic data thus suggest that at low temperatures the odd-numbered methyl anteisobranched phosphatidylcholines form a highly ordered condensed phase similar to the Lc phases of the linear saturated n-acyl-phosphatidylcholines. A comparable condensed phase was not formed by the even-numbered anteisobranched phosphatidylcholines under similar conditions. The properties of the gel states of the even-numbered anteisoacylphosphatidylcholines were generally similar to those of the high-temperature gel states of their odd-numbered counterparts. Those gel states exhibit spectral characteristics indicative of hexagonally packed but relatively mobile acyl chains. The temperature-dependent changes in the spectral characteristics of these gel states were continuous and were not resolved into the discrete but overlapping transitions observed by differential scanning calorimetry.     

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains.     

Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains

The structure of the lectin discoidin I has been studied by circular dichroism and fluorescence spectroscopy. A positive ellipticity band at 224 nm is detected in the CD spectrum of discoidin I. The fluorescence spectra show a defined shoulder at 325 nm that through acrylamide quenching has been associated with a displaced tryptophan residue partly buried in the discoidin I molecule. This tryptophan could also be responsible for the 224 nm positive band of the CD spectrum. These spectroscopic characteristics of discoidin I indicate the existence of structural homologies with fibronectin, where the optical activity of aromatic chromophores has been associated with the positive ellipticity band at 227 nm. The CD adjust parameters and theoretical secondary structure predictions show that discoidin I is a molecule with a low content of alpha-helix and beta-strand and high content of beta-turn structures, similar to other lectins.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 2. Infrared and 31P NMR spectroscopic studies

The polymorphic phase behavior of aqueous dispersions of a number of representative phosphatidylcholines with methyl iso-branched fatty acyl chains was investigated by Fourier transform infrared (FT-IR) and phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy. For the longer chain phosphatidylcholines, where two transitions are resolved on the temperature scale, the higher temperature event can unequivocally be assigned to the melting of the acyl chains (i.e., a gel/liquid-crystalline phase transition), whereas the lower temperature event is shown to involve a change in the packing mode of the methylene and carbonyl groups of the hydrocarbon chains in the gel state (i.e., a gel/gel transition). The infrared spectroscopic data suggest that the methyl iso-branched phosphatidylcholines assume a partially dehydrated, highly ordered state at low temperatures, resembling the Lc phase recently described for the long-chain n-saturated phosphatidylcholines. At higher temperatures, some branched-chain phosphatidylcholines appear to assume a fully hydrated, loosely packed gel phase similar to but not identical with the P beta, phase of their linear saturated analogues. Thus, the iso-branched phosphatidylcholine gel/gel transition corresponds, at least approximately, to a summation of the structural changes accompanying both the subtransition and the pretransition characteristic of the longer chain n-saturated phosphatidylcholines. The infrared spectroscopic data also show that, in the low-temperature gel state, there are significant differences between the odd- and even-numbered isoacylphosphatidylcholines with respect to their hydrocarbon chain packing modes as well as to their head group and interfacial hydration states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipid's chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.     

A calorimetric examination of the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes

The effect of myotoxin a on the thermotropic phase behavior of aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was examined using differential scanning calorimetry (DSC). Myotoxin a significantly altered the normal phase behavior of DMPC in a concentration dependent fashion. This effect is perturbed by Ca2+ and is sensitive to ionic strength and pH. High concentrations of toxin eliminate the characteristic pretransition associated with the polar head group of DMPC. They also increase the temperature of the main gel-to-liquid crystal transition from 23 degrees C to 32-35 degrees C. At low concentrations of toxin, the first visible effect is upon the pretransition which is split into two components that diminish with time. The main transition is less affected at low toxin concentrations, although the magnitude of the transition is reduced while it is simultaneously shifted to higher temperatures. The main transition is also split into multiple components. The toxin also had pH specific effects on the phase behavior of DMPS. Above physiological pH (8.5) the normal transition of DMPS at 36-38 degrees C was split in the presence of myotoxin a and new components appeared centered at 31 degrees C and 35 degrees C. These observations are consistent with reports that the skeletal muscle membrane system is the major site of the myonecrotic effect of myotoxin a.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes

We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

The effect of hydrostatic pressure on the bilayer structure of phosphatidylcholines containing omega-cyclohexyl fatty acyl chains

The barotropic behavior of aqueous dispersions of two representative omega-cyclohexyl phosphatidylcholines was investigated by pressure-tuning Fourier transform infrared spectroscopy. In the even-numbered homologue, 1,2-di-14-cyclohexyltetradecanoyl-sn-glycero-3-phosphocholine (14cyPC), the lipid molecules are orientationally disordered until the applied pressure reaches 2.1 kbar. This pressure marks the onset of correlation field splitting of the scissoring and rocking modes of the linear chain methylenes, as well as that of the cyclohexyl ring methylenes. It indicates immobilization of the entire acyl chains, whereby the zig-zag planes of the neighboring straight chain all-trans methylenes are oriented mainly perpendicular to each other. As judged from the magnitude of the correlation field splittings, the interchain interaction is weaker in 14cyPC than that in linear lipids (e.g., DMPC or DPPC). Upon an increase in pressure, up to 20 kbar, the zig-zag methylene planes in 14cyPC undergo a gradual transformation to a parallel orientation. In the odd-numbered homologue, 1,2-di-13-cyclohexyltridecanoyl-sn-glycero-3-phosphocholine (13cyPC), there is no correlation field splitting originating from the straight chain methylenes (up to 21 kbar). The linear, nonbranched segments of the omega-cyclohexyl chains in 13cyPC are closely packed with the all-trans methylene zig-zag planes oriented parallel to each other. There is, however, correlation field splitting of the ring methylenes, indicating interring interactions between the bulky cyclohexyl rings in opposing bilayer leaflets. There are major structural differences between the even- and odd-numbered homologues in the interfacial region, which remain even at high pressures. The ester carbonyl C = O stretching band in 14cyPC is a composite of two discrete bands which do not change considerably in intensity or frequency in the pressure range 2-20 kbar. In contrast, 13cyPC possesses an additional, low-frequency C = O stretching component at low pressures. As the pressure increases, the three component bands coalesce into a single C = O stretching band. Our results suggest equally oriented, fully hydrogen-bonded carbonyl groups in 13cyPC at pressures above approx. 10 kbar.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

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

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)     

A differential scanning calorimetric study of brain clathrin

The thermal denaturation of clathrin-coated vesicles isolated from bovine brain tissue has been studied by differential scanning calorimetry and has been compared to basket structures reformed from isolated triskelion trimers of clathrin and to isolated triskelions. The coated vesicles and reformed baskets displayed similar, yet distinct, thermal behavior. Calorimetric data of the coated vesicles exhibited a single denaturation transition peak at 55.9 +/- 0.1 degrees C, skewed to low temperatures whereas the thermograms for the reformed baskets exhibited a broad transition peak at 53.1 +/- 0.1 degrees C and a peak at 56.3 +/- 0.1 degrees C. Neither transition was reversible. The specific transition enthalpy was 11.5 +/- 1.0 J g-1 for the coated vesicles and the total transition enthalpy was 9.1 +/- 0.3 J g-1 for the reformed baskets. In contrast, isolated triskelions showed no thermal transition between 15 and 90 degrees C. Although the coated vesicles and the reformed baskets have similar stability reflecting their similar structures, the coated vesicles appear to be marginally more stable than the reformed baskets. The complexity of the transition profiles and their lack of symmetry suggest the existence of several, somewhat independent, domains unique to the cage-like structure of the coated vesicles and reformed baskets.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.