Non-interdigitated Gel Phase Research Articles

Vesicle Origami and the Influence of Cholesterol on Lipid Packing.

The artificial phospholipid Pad-PC-Pad was analyzed in 2D (monolayers at the air/water interface) and 3D (aqueous lipid dispersions) systems. In the gel phase, the two leaflets of a Pad-PC-Pad bilayer interdigitate completely, and the hydrophobic bilayer region has a thickness comparable to the length of a single phospholipid acyl chain. This leads to a stiff membrane with no spontaneous curvature. Forced into a vesicular structure, Pad-PC-Pad has faceted geometry, and in its extreme form, tetrahedral vesicles were found as predicted a decade ago. Above the main transition temperature, a noninterdigitated Lα phase with fluid chains has been observed. The addition of cholesterol leads to a slight decrease of the main transition temperature and a gradual decrease in the transition enthalpy until the transition vanishes at 40 mol % cholesterol in the mixture. Additionally, cholesterol pulls the chains apart, and a noninterdigitated gel phase is observed. In monolayers, cholesterol has an ordering effect on liquid-expanded phases and disorders condensed phases. The wavenumbers of the methylene stretching vibration indicate the formation of a liquid-ordered phase in mixtures with 40 mol % cholesterol.

Effects of cholesterol on phospholipid membranes: Inhibition of the interdigitated gel phase of F-DPPC and F-DPPC/DPPC

Unlike the parent phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the monofluorinated analog, 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC), spontaneously forms an interdigitated gel phase (LβI) below the main transition temperature (Tm). We have examined the effects of introducing cholesterol to F-DPPC and 1:1 F-DPPC/DPPC membranes using a combination of DSC, optical density, fluorescence intensity and polarization, 31P NMR, and X-ray diffraction techniques. Cholesterol increases the fluidity of the gel phase, broadens the main transition, and decreases the main transition enthalpy. However, these results also reveal that there is an unusually large degree of phase coexistence between the LβI and non-interdigitated gel phases when cholesterol is added. Cholesterol encourages this phase segregation by partitioning into the thicker non-interdigitated domains. At higher cholesterol concentrations, the majority or all of the LβI phase of F-DPPC and 1:1 F-DPPC/DPPC is eliminated and is replaced by a non-interdigitated liquid-ordered (lo) phase with properties similar to DPPC/cholesterol. Consequently, cholesterol mitigates the influence the CF moiety has on the thermodynamic phase behavior of F-DPPC. Our findings demonstrate that there are multiple characteristics of cholesterol-rich membranes that disfavor interdigitation.

Pressure induces interdigitation differently in DPPC and DPPG

The phase behaviours of chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62) and chain-perdeuterated dipalmitoylphosphatidylglycerol (DPPG-d62) bilayers were compared using 2H nuclear magnetic resonance spectroscopy and quadrupole echo decay measurements over pressures ranging from ambient to 196 MPa and temperatures ranging from 60 to -25 degrees C. At ambient pressure, the phase behaviours of DPPC-d62 and DPPG-d62 were nearly identical. At 196 MPa, their behaviours were also very similar and both lipids appeared to pass from an interdigitated gel phase at high temperature, through a non-interdigitated gel phase at intermediate temperature, to a chain-immobilized ordered phase at low temperature. At 85 MPa, the behaviour of DPPC-d62 was similar to its ambient pressure behaviour with no evidence of interdigitation. For DPPG-d62, however, the behaviour at 85 MPa was similar to its higher pressure behaviour and spectra characteristic of an interdigitated gel phase were observed. Pressure-temperature phase diagrams for both lipids were compared. While the minimum pressure for DPPC-d62 interdigitation is about 150 MPa, DPPG-d62 was observed to interdigitate at pressures as low as 60 MPa. Given the similarity of their phase behaviours at both higher and lower pressures, this difference reflects the extent to which bilayer phase behaviour depends on the balance between interactions in the headgroup and hydrocarbon regions of the bilayer.

Studies of the Structure and Organization of Cationic Lipid Bilayer Membranes: Calorimetric, Spectroscopic, and X-Ray Diffraction Studies of Linear Saturated P-O-Ethyl Phosphatidylcholines

Differential scanning calorimetry, x-ray diffraction, and infrared and 31P-nuclear magnetic resonance ( 31P-NMR) spectroscopy were used to examine the thermotropic phase behavior and organization of cationic model membranes composed of the P-O-ethyl esters of a homologous series of n-saturated 1,2-diacyl phosphatidylcholines (Et-PCs). Differential scanning calorimetry studies indicate that on heating, these lipids exhibit single highly energetic and cooperative endothermic transitions whose temperatures and enthalpies are higher than those of the corresponding phosphatidylcholines (PCs). Upon cooling, these Et-PCs exhibit two exothermic transitions at temperatures slightly below the single endotherm observed upon heating. These cooling exotherms have both been assigned to transitions between the liquid-crystalline and gel phases of these lipids by x-ray diffraction. The x-ray diffraction data also show that unlike the parent PCs, the chain-melting phase transition of these Et-PCs involves a direct transformation of a chain-interdigitated gel phase to the lamellar liquid-crystalline phase for the homologous series of n ≥ 14. Our 31P-NMR spectroscopic studies indicate that the rates of phosphate headgroup reorientation in both gel and liquid-crystalline phases of these lipids are comparable to those of the corresponding PC bilayers. However, the shape of the 31P-NMR spectra observed in the interdigitated gel phase indicates that phosphate headgroup reorientation is subject to constraints that are not encountered in the non-interdigitated gel phases of parent PCs. The infrared spectroscopic data indicate that the Et-PCs adopt a very compact form of hydrocarbon chain packing in the interdigitated gel phase and that the polar/apolar interfacial regions of these bilayers are less hydrated than those of corresponding PC bilayers in both the gel and liquid-crystalline phases. Our results indicate that esterification of PC phosphate headgroups results in many alterations of bilayer physical properties aside from the endowment of a positively charged surface. This fact should be considered in assessing the interactions of these compounds with naturally occurring lipids and with other biological materials.

A Macroscopic Description of Lipid Bilayer Phase Transitions of Mixed-Chain Phosphatidylcholines: Chain-Length and Chain-Asymmetry Dependence

A macroscopic model is presented to quantitatively describe lipid bilayer gel to fluid phase transitions. In this model, the Gibbs potential of the lipid bilayer is expressed in terms of a single order parameter q, the average chain orientational order parameter. The Gibbs potential is based on molecular mean-field and statistical mechanical calculations of inter and intrachain interactions. Chain-length and chain-asymmetry are incorporated into the Gibbs potential so that one equation provides an accurate description of mixed-chain phosphatidylcholines of a single class. Two general classes of lipids are studied in this work: lipid bilayers of partially or noninterdigitated gel phases, and bilayers of mixed interdigitated gel phases. The model parameters are obtained by fitting the transition temperature and enthalpy data of phosphatidylcholines to the model. The proposed model provides estimates for the transition temperature and enthalpy, van der Waals energy, number of gauche bonds, chain orientational order parameter, and bond rotational and excluded volume entropies, achieving excellent agreement with existing data obtained with various techniques.

Preferential Interactions of Fluorescent Probe Prodan with Cholesterol

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal L α , 435 nm for the gel L′ β , and 507 nm for the interdigitated gel L β I phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 × 10 4 in the interdigitated gel, 1.8 × 10 4 in the noninterdigitated gel, and 7.6 × 10 4 in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 × 10 4 for the liquid crystal and the gel phase, and 5.1 × 10 4 in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.

Phase transition sequence between fluid liquid-crystalline and interdigitated lamellar gel phases in mixed-chain diacyl phosphatidylcholine

The phase transition behavior of asymmetric phosphatidycholine with palmitic acid at the sn-1 position and capric acid at the sn-2 position (C16C10PC) has been examined by simultaneous differential scanning calorimetry and synchrotron X-ray diffraction complemented by electron spin resonance. The experiments were performed for a sequence of cooling, heating and recooling scans of aqueous dispersions of C16C10PC. The results showed that a single exothermic peak centered at 5°C appeared on the initial cooling. It coincided with a transition of a liquid-crystalline lamellar phase (d-spacing, 6.4 nm) directly into an interdigitated lamellar gel phase (d-spacing, 5.2 nm). An immediate reheating scan showed a single broad endothermic transition peak in the temperature range 3–10°C over which there was a direct transition from the interdigitated lamellar gel phase to the fluid liquid-crystalline lamellar phase with coexistence of the two phases in the region of the transition. Coexistence of phases was also inferred from ESR evidencing two different environments for 16 doxyl stearic acid probe. Unlike non-interdigitated gel phases of symmetric phospholipids, the interdigitated gel phase of C16C10PC is typified by a high value of the magnetic hyperfine coupling constant. This constant (16 Gauss) indicates around the nitroxide group a viscous highly ordered environment with hydrogen bonding within the interdigitated bilayer. The subsequent cooling scan was markedly different from the initial cooling scan since the thermogram showed two exothermic peaks. The first peak at 7°C, representing 30% of the overall enthalpy change, coincided with the conversion of fluid liquid-crystalline phase into non-interdigitated lamellar gel phase (d-spacing, 6.32 nm). The second peak at 5°C is observed when the interdigitated lamellar gel phase (d-spacing, 5.2 nm) takes place. This behavior is consistent with the buildup of extended lamellar sheets with low curvature induced by cooling down vesicles with high radius of curvature fusing when the interdigitated lamellar gel phase takes place beyond the transition temperature. Time-resolved DSC and X-ray diffraction show differences with ESR at the transition temperature which support the occurrence of a metastable non-interdigitated gel between the fluid and the interdigitated lamellar gel in the transition sequence.

Greater partitioning of small spin labels into interdigitated than into non-interdigitated gel phase bilayers

Partitioning of the small amphipathic spin labels 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-oxo-2,2,6,6-tetramethylpiperidinyl- N-oxy (TEMPONE), and di-tertiary butyl nitroxide (DTBN) into interdigitated L β I and non-interdigitated L β ′, P β ′, and L α phases was compared. The double chain interdigitated phases examined were 1,2-dipalmitoylphosphatidylcholine (DPPC) in the presence of 2.17 M ethanol, dihexadecylphosphatidylcholine (DHPC), and the complexes of dipalmitoylphosphatidylglycerol (DPPG) with polymyxin B and myelin basic protein. The triple chain mixed interdigitated phase of the asymmetric chain 1-stearoyl-2-caproyl-phosphatidylcholine (18:10-PC) was also examined. The non-interdigitated lipids were DPPC, DPPC in the presence of 0.87 M ethanol, DPPG, and the complex of DPPG with polylysine. Quantitation of the percentage of the membrane-bound spectral contribution in the presence of NiSO 4, used to broaden the aqueous spectral contribution, showed that all three spin labels partitioned into the L β I phase significantly more than into the L β ′ phase. The hyperfine splitting values indicated that all three spin labels were located near the apolar/polar interface in the L β I phase. The amount of TEMPO and DTBN bound to the L β I phase was similar to that in the L α phase but more TEMPONE was bound to the L β I phase than to the L α phase. This is attributed to the greater polarity of TEMPONE, its preference for a polar or amphipathic environment, and the larger volume between lipid head groups at the apolar/polar interface in the L β I phase compared to the other phases. However, TEMPO binding to the mixed interdigitated gel phase of 18:10-PC was no greater than to the non-interdigitated L β ′ phase of DPPC, reflecting the smaller volume at the apolar/polar interface of this triple chain interdigitated bilayer compared to the double chain interdigitated bilayers. Thus interdigitation of the lipid bilayer, whether due to addition of a substance like ethanol, or spontaneous as in DHPC, can cause increased partitioning of other substances into the bilayer, provided that they are small enough to fit into the volume created at the apolar/polar interface. This increased partitioning could contribute to an increased permeability of small molecules across interdigitated bilayers.

Lipid chain motion in an interdigitated gel phase: conventional and saturation transfer ESR of spin-labeled lipids in dipalmitoylphosphatidylcholine-glycerol dispersions.

The lipid chain dynamics in the interdigitated gel phase of dipalmitoylphosphatidylcholine (DPPC) dispersed in glycerol and in the fully hydrated noninterdigitated gel phase in aqueous buffer were compared by using conventional and saturation transfer electron spin resonance (ESR) spectroscopy. Twelve different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain were used to characterize the chain motion. The outer hyperfine splittings of the conventional ESR spectra and the line height ratios at the diagnostic spectral positions in the saturation transfer ESR spectra were taken as indices of the rotational mobility of the labeled chain segments in the gel phase (0-40 degrees C). The conventional spin label ESR spectra revealed a gradient of increasing mobility on proceeding down the chain toward the terminal methyl end in the fully hydrated DPPC gel phase bilayer structure. This gradient was absent in the interdigitated gel phase, i.e., the rotational mobility throughout the length of the lipid chain was comparable to that near the polar interface, on the conventional ESR time scale. Values of the outer hyperfine splitting for spin labels at the 5- and 14-C atom positions in the chain were 65.5 and 61.0 G in buffer, respectively, and 67.0 G for both positions in glycerol, at 0 degrees C. At 35 degrees C, still in the gel phase, these differences between the two systems were much greater. Saturation transfer ESR measurements revealed that the motion throughout the chain was restricted on the microsecond time scale in the interdigitated phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Interdigitated gel phase bilayers formed by unsaturated synthetic and bacterial glycerolipids in the presence of polymyxin B and glycerol

The ability of synthetic phosphoglycerolipids with a cis mono-unsaturated acyl chain in the 2-position and a saturated chain in the 1-position of glycerol to form interdigitated gel phase bilayers in the presence of amphipathic substances was monitored using a fatty acid spin label, 16-doxylstearic acid, and phosphatidylglycerol spin label containing 16-doxylstcaric acid. These spin labels become significantly more motionally restricted in an interdigitated gel phase bilayer than in a non-interdigitated gel phase bilayer. The results indicated that polymyxin B and polymyxin B nonapeptide caused interdigitation of 1-palmitoyl,2-oleoyl-phosphatidylglycerol (POPG) and glycerol caused interdigitation of 1-stearoyl,2-olcoyl-phosphatidylcholine (SOPC), similar to their effects on disaturated lipids. The fluidity gradient present in non-interdigitated gel phase bilayers was abolished. However, glycerol did not cause POPG to become interdigitated, in contrast to SOPC. We reported earlier that there is a kinetic barrier to interdigitation of saturated PG in the presence of glycerol, in contrast to saturated PC. This barrier is even greater for the unsaturated species of PG. Furthermore, these compounds lowered the gel to liquid-crystalline phase transition temperatures of the unsaturated lipids more than of saturated lipids suggesting that the interdigitated bilayer of the former may be less ordered or less stable than that of the latter. Since polymyxin B is an antibiotic we also examined its effect on a lipid extract from the Gram-negative bacteria Pseudomonas aeruginosa in order to assess whether interdigitation might be involved in its mechanism of bactericidal or bacteriostatic effect. Polymyxin B and polymyxin B nonapeptide also caused motional restriction of a small percentage (about 13% at −2°C and 25% at −14°C for polymyxin B) of the spin label in the lipid extract at low temperatures, where the lipid is in the gel phase, consistent with formation of a small domain of interdigitated bilayer lipid. However, the degree of immobilization was less than that in the interdigitated bilayers of the synthetic unsaturated lipids. This may be a result of the heterogeneous nature of the lipids in the extract. However, it cannot be ruled out that the motional restriction of the spin label in this extract may be caused by something other than interdigitation. Thus the results with the lipid extract are less conclusive of interdigitation than for the synthetic lipids. A motionally restricted population was not detectable at higher temperatures. This study shows that a cis-double bond in the acyl chain of phosphoglycerolipids does not prevent formation of an interdigitated gel phase bilayer. However, such lipids are in the liquid-crystalline phase at physiological temperature and thus will not form interdigitated domains in biological membranes.

Thermotropic properties of saturated mixed acyl phosphatidylethanolamines

The mixed acyl phosphatidylethanolamine (PE) series C(18)C(18)PE, C(18)C(16)PE, C(18)C(14)PE, C(18)C(12)PE, and C(18)C(10)PE has been prepared from the corresponding phosphatidylcholines by phospholipase D mediated transphosphatidylation. The thermotropic behavior of unhydrated and hydrated preparations of these PEs has been investigated by differential scanning calorimetry and 31P NMR spectroscopy. Unhydrated preparations of the PEs undergo crystalline to liquid-crystalline transitions (Tm+h), which correspond to the simultaneous hydration and acyl chain melting of poorly hydrated crystalline samples. Hydrated preparations of the PEs undergo gel to liquid-crystalline transitions (Tm) when scanned immediately subsequent to cooling from temperatures above their respective Tm+hs. Multilamellar bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE pack without significant interdigitation of the phospholipid acyl chains across the bilayer center in the gel phase. C(18)C(10)PE multilamellar preparations exhibit a mixed-interdigitated gel phase packing of the phospholipid acyl chains. Hydrated bilayers of C(18)C(12)PE adopt a mixed-interdigitated gel phase packing at temperatures below 13.9 degrees C. Between 13.9 degrees C and the gel to liquid-crystalline transition temperature of 36.9 degrees C, the C(18)C(12)PE bilayer adopts a noninterdigitated gel phase packing. The metastable behavior of fully hydrated and partially hydrated preparations of the mixed acyl PEs has been investigated. Bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE exhibited little or no tendency toward regeneration of the crystalline phase. In contrast, bilayers of C(18C(12)PE and C(18)C(10)PE exhibited a metastability of the liquid-crystalline phase in the temperature interval between Tm and Tm+h, which can allow for the regeneration of the crystalline phase under certain conditions. Bilayers of C(18)C(12)PE exhibited an additional metastability of the noninterdigitated gel phase.

Solvent-mediated alterations of the stratum corneum

The intercellular lipid multilayers of stratum corneum, the outermost layer of mammalian skin, are generally accepted as the rate-determining pathway of transmembrane diffusion for lipophilic solutes. The effects of a series of saturated and unsaturated octadecanoic acids (saturated stearic acid, cis- and trans-octadecenoic acid) and a series of short-chain alcohols (ethanol, n-propanol, isopropanol and n-butanol) on the thermotropic phase behavior of stratum corneum have been investigated. In conjunction with the thermotropic phase behavior, fatty-acid-induced effects on the permeation of a lipophilic compound have also been studied. The alcohol-induced effects were determined at concentrations that haue given penetration enhancement for the respective alcohols in order to relate the findings to available skin transport data from literature. Permeation enhancement of lipophilic solutes does not necessarily occur with a concomitant decrease in the gel to liquid-crystalline phase transition. The unsaturated fatty acids in the presence of aqueous ethanol solutions have been associated with conformational alterations of the gel phase lipid domain. However, the effect of fatty acids is complicated by the presence of the cosolvent. Therefore, the thermotropic phase behavior of the stratum corneum was also investigated in the presence of a series of short-chain alcohols. Short-chain alcohol enhanced permeation may result from a reduction of lipid polar head interactions, an inter facial transport pathway between limited populations of interdigitated and noninterdigitated gel phases within the membrane or further disordering of possible liquid-crystalline phases within the membrane.

The ordering and dynamics of the terminal methyl group on the lignoceric acid chain in cerebroside sulfate

Cerebroside sulfate containing lignoceric acid deuterated at the terminal methyl group was prepared by a semi-synthetic method in order to study the ordering and motion of the fatty acyl chain by deuterium NMR spectroscopy. The lipid was hydrated with either 2 M KCl or 2 M LiCl. The NMR results are consistent with the formation of an interdigitated bilayer in the gel phase as suggested by spin label and X-ray diffraction studies, although they do not delineate whether the interdigitation is of the partial or mixed triple-chain type. In the low-temperature stable phase formed by this lipid on slow cooling from the liquid-crystalline phase, which has been shown by X-ray diffraction to be partially interdigitated, the dominant molecular motion is reorientation of the lipid about its along axis. The motion had a correlation time of the order of the deuterium quadrupole coupling constant (10 −6 s). The rate of axial diffusion is greater than that found for symmetric phospho- or glycosphingo-lipids in their crystalline subgel phases, but less than that of a lipid which forms a non-interdigitated gel phase bilayer such as DPPE, or a lipid which forms a fully interdigitated gel phase bilayer like DHPC. The rate of motion appears to be greater in a low temperature, more hydrated metastable phase formed by this lipid after cooling rapidly from the liquid crystalline phase. This phase is thought to be a mixed interdigitated bilayer based on spin label studies. The disorder observed at the C-terminal methyl group in the L α phase, which is probably partially interdigitated according to X-ray diffraction studies, is greater than that of a non-interdigitated bilayer formed by a symmetric lipid. In the CBS/Li + system at high temperature, there is evidence for a liquid-crystalline to liquid-crystalline phase transition, in which the average orientation of the terminal methyl group changes.

Use of spin labels to determine the percentage of interdigitated lipid in complexes with polymyxin B and polymyxin B nonapeptide

Long chain spin labels with the nitroxide group located near the terminal methyl of the chain were used to determine the percentage interdigitated lipid in complexes of polymyxin B (PMB) and polymyxin B nonapeptide (PMBN) with the acidic lipids dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidic acid (DPPA) at varying mole ratios of drug to lipid and at different pH values. These spin labels are more motionally restricted in the interdigitated than in the non-interdigitated gel phase bilayer. This allows determination of the percentage interdigitated lipid by resolution of the spectrum into motionally restricted and more mobile components. At nonsaturating concentrations of PMB, significantly more DPPG than that which can be maximally PMB-bound, becomes interdigitated. As the temperature approaches the gel to liquid crystalline phase transition temperature, the bilayer becomes progressively non-interdigitated. The ESR spectrum indicates that PMB also causes interdigitation of DPPA. However, in contrast to DPPG, the amount of DPPA which is interdigitated at pH 6, is less than the amount which is expected to be PMB-bound. This is attributed to the ability of DPPA to participate in lateral interlipid hydrogen bonding interactions. Such lateral interactions would be abolished in the interdigitated bilayer and thus they are expected to inhibits its formation. At pH 9, where the interlipid interactions of DPPA are weakened, PMB induces even more lipid than that which is PMB-bound to become interdigitated. Indeed, the percentage interdigitated lipid is even greater than found for DPPG. This may be partly a result of the greater negative charge of DPPA at this pH. A greater repulsive negative charge is expected to favor interdigitation. PMBN is less effective than PMB at inducing interdigitation of DPPG and causes little or no interdigitation of DPPA at pH 6, even at saturating concentrations. PMBN also does not lower the phase transition temperature of DPPA at pH 6 as much as PMB. At pH 9, the effect of PMBN on DPPA is more similar to the effect of PMB. However, even for DPPG, and DPPA at pH 9, PMBN does not maintain interdigitation of the lipids at higher temperatures as effectively as PMB. PMBN's smaller perturbing effect and greatly decreased ability to cause interdigitation of DPPA at pH values below 9 may be related to a decreased ability to cause lateral separation of the lipid molecules, which is necessary in order to weaken the interlipid interactions. PMBN lacks the fatty acyl chain of PMB, which may help cause lateral separation of the lipid molecules. These results may be related to the differences in physiological effects of the antibiotic PMB and the nonbactericidal PMBN on Gram-negative bacteria.

Behavior of spin labels in a variety of interdigitated lipid bilayers

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dipalmitoylphosphatidycholine /glycerol, dipalmitoylphosphatidylglycerol/polymyxin B, and the asymmetric 1-stearoyl-2-caproylphosphatidylcholine. A difference can also be detected for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with my spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.

Mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers: evidence for gel and liquid-crystalline phase immiscibility.

The mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers has been investigated by high-sensitivity differential scanning calorimetry. Phase diagrams have been constructed for two-component bilayers composed of C(18)C(18)PC and either C(18)C(16)PC, C(18)C(14)PC, C(18)C(12)PC, or C(18)C(10)PC. It is found that C(18)C(18)PC-C(18)C(16)PC and C(18)C(18)PC-C(18)C(14)PC mixed bilayers exhibit complete miscibility of the components in both the gel and liquid-crystalline phases. Whereas this mixing is observed to be nearly ideal for the C(18)C(18)PC-C(18)C(16)PC binary system, the intermixing of the lipids is highly nonideal in the gel phase of the C(18)C(18)PC-C(18)C(14)PC binary mixture. The C(18)C(18)PC-C(18)C(12)PC and C(18)C(18)PC-C(18)C(10)PC mixed bilayers are characterized by partial immiscibility of the phosphatidylcholine components in the bilayer gel phase. Over a large compositional range, these bilayers appear to consist of phase-separated regions of interdigitated and noninterdigitated gel phases. In addition, the C(18)C(18)PC-C(18)C(10)PC two-component bilayer displays a limited region of liquid-liquid immiscibility in the liquid-crystalline bilayer phase. The phase separation of the mixed chain length phosphatidylcholines revealed in these mixed bilayers may represent a three-dimensional phase separation of the lipid components where the phosphatidylcholines are both laterally separated within the plane of the bilayer and conformationally coupled across the bilayer. Such phase-separated domains could have profound effects on membrane structure and function if they were to occur in biological membranes.

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidyglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97–108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175–178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.