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.     

Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol (Chol) and epicholesterol (EChol) on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine (DPPC) bilayers. EChol is an epimer of Chol in which the axially oriented hydroxyl group of C3 of Chol is replaced by an equatorially oriented hydroxyl group, resulting in a different orientation of the hydroxyl group relative to sterol fused ring system. Our calorimetric studies indicate that the incorporation of EChol is more effective than Chol is in reducing the enthalpy of the pretransition of DPPC. EChol is also initially more effective than Chol in reducing the enthalpies of both the sharp and broad components of the main phase transition of DPPC. However, at higher EChol concentrations (~ 30-50 mol%), EChol becomes less effective than Chol in reducing the enthalpy and cooperativity of the main phase transition, such that at sterol concentrations of 50 mol%, EChol does not completely abolish the cooperative hydrocarbon chain-melting phase transition of DPPC, while Chol does. However, EChol does not appear to form a calorimetrically detectable crystallite phase at higher sterol concentrations, suggesting that EChol, unlike Chol, may form dimers or lower order aggregates at higher sterol concentrations. Our spectroscopic studies demonstrate that EChol incorporation produces more ordered gel and comparably ordered liquid-crystalline bilayers compared to Chol, which are characterized by increased hydrogen bonding in the glycerol backbone region of the DPPC bilayer. These and other results indicate that monomeric EChol is less miscible in DPPC bilayers than is Chol at higher sterol concentrations, but perturbs their organization to a greater extent at lower sterol concentrations, probably due primarily to the larger effective cross-sectional area of the EChol molecule. Nevertheless, EChol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers.     

A comparative calorimetric study of the effects of cholesterol and the plant sterols campesterol and brassicasterol on the thermotropic phase behavior of dipalmitoylphosphatidylcholine bilayer membranes

We present a comparative differential scanning calorimetric study of the effects of the animal sterol cholesterol (Chol) and the plant sterols campesterol (Camp) and brassicasterol (Bras) on the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers. Camp and Bras differ from Chol in having a C24 methyl group and, additionally for Bras, a C22 trans-double bond. Camp and especially Bras decrease the temperature, cooperativity and enthalpy of the DPPC pretransition more than Chol, although these effects are attenuated at higher sterol levels. This indicates that they destabilize gel-state DPPC bilayers to a greater extent, but are less soluble, than Chol. Not surprisingly, all three sterols have similar effects on the sterol-poor sharp component of the DPPC main phase transition. However, Camp and especially Bras less effectively increase the temperature and decrease the cooperativity and enthalpy of the broad component of the main transition than Chol. This indicates that at higher sterol concentrations, Camp and Bras are less miscible and less effective than Chol at ordering the hydrocarbon chains of the sterol-enriched fluid DPPC bilayers. Overall, these alkyl side chain modifications generally reduce the ability of Chol to produce its characteristic effects on DPPC bilayer physical properties. These differences are likely due to the less extended and more bent conformations of the alkyl side chains of Camp and Bras, producing sterols with a greater effective cross-sectional area and reduced length than Chol. Hence, the structure of Chol is likely optimized for maximum solubility in, as opposed to maximum ordering of, phospholipid bilayers.     

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and ³¹P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner (Biochemistry 1993, 32:516-522), but is in contrast to our previous results for phosphatidylethanolamine (Biochim. Biophys. Acta 1999, 1416:119-234) and phosphatidylserine (Biophys. J. 2000, 79:2056-2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids 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.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the “left endpoint” and “right endpoint” of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

A detailed analysis of partial molecular volumes in DPPC/cholesterol binary bilayers

We examined the volumetric behavior of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol binary bilayer system with high accuracy and more cholesterol concentrations to reveal the detailed molecular states in the liquid-disordered (L_d) phase, the liquid-ordered (L_o) phase and the gel phase. We measured the average specific volume of the binary bilayer at several temperatures by the neutral flotation method and calculated the average volume per molecule to estimate the partial molecular volumes of DPPC and cholesterol in each phase. As a result, we found that the region with intermediate cholesterol concentrations showed a more complicated behavior than expected from simple coexistence of L_d and L_o domains. We also measured fluorescence decay of trans-parinaric acid (tPA) added into the binary bilayer with more cholesterol concentrations to get further insight into the cholesterol-induced formation of the L_o phase. On the basis of these results we discuss the molecular interaction between DPPC and cholesterol molecule in the L_o phase and the manner of L_d/L_o phase coexistence.     

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

We performed differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopic measurements to study the effects of lathosterol (Lath) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes and compared our results with those previously reported for cholesterol (Chol)/DPPC binary mixtures. Lath is the penultimate intermediate in the biosynthesis of Chol in the Kandutsch-Russell pathway and differs from Chol only in the double bond position in ring B, which is between C7 and C8 in Lath and between C5 and C6 in Chol. Our DSC studies indicate that the incorporation of Lath is more effective than Chol in reducing the temperature and enthalpy of the DPPC pretransition. At lower sterol concentrations (≤10 mol %), incorporation of both Lath and Chol decreases the temperature, enthalpy, and cooperativity of the sharp component of the main phase transition of DPPC to a similar extent, but at higher sterol concentrations, Lath is more effective at decreasing the phase transition temperature, enthalpy, and cooperativity than Chol. These results indicate that at higher concentrations, Lath is more disruptive of DPPC gel-state bilayer packing than Chol is. Moreover, incorporation of Lath decreases the temperature of the broad component of the main phase transition of DPPC, whereas Chol increases it; this difference in the direction and magnitude of the temperature shift is accentuated at higher sterol concentrations. Although at sterol concentrations of ≤20 mol % Lath and Chol are almost equally effective at reducing the enthalpy and cooperativity of the broad component of the main phase transition, at higher sterol levels Lath is less effective than Chol in these regards and does not completely abolish the cooperative hydrocarbon chain melting phase transition at 50 mol %, as does Chol. These latter results indicate that Lath both is more disruptive with respect to the low-temperature state of the sterol-enriched domains of DPPC bilayers and has a lower lateral miscibility in DPPC bilayers than Chol. Our FTIR spectroscopic studies suggest that Lath incorporation produces a less tightly packed bilayer than does Chol at both low (gel state) and high (liquid-crystalline state) temperatures, which is characterized by increased H-bonding between water and the carbonyl groups of the fatty acyl chains in the DPPC bilayer. Overall, our studies indicate that Lath and Chol incorporation can have rather different effects on the thermotropic phase behavior and organization of DPPC bilayers and thus that the position of the double bond in ring B of a sterol molecule can have an appreciable effect on the physical properties of sterol molecules.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with d-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Interactions of the dipeptide paralysin β-Ala-Tyr and the aminoacid Glu with phospholipid bilayers

Existing evidence points out that the biological activity of β-Ala-Tyr may in part related to its interactions with the cell membranes. For comparative reasons the effects of Glu were also examined using identical techniques and conditions. In order to examine their thermal and dynamic effects on membrane bilayers a combination of DSC, Raman and solid state NMR spectroscopy on DPPC/water model membranes were applied and the results were compared. DSC data showed that Glu perturbs to a greater degree the model membrane compared to β-Ala-Tyr. Thus, alteration of the phase transition temperature and half width of the peaks, abolishment of the pretransition and influence on the enthalpy of the phase transition were more pronounced in the Glu loaded bilayers. Raman spectroscopy showed that incorporation of Glu in DPPC/water bilayers increased the order in the bilayers in contrast to the effect of the dipeptide. Several structural and dynamical properties of the DPPC multilamellar bilayers with and without the dipeptide or Glu were compared using high resolution C-13 MAS (Magic Angle Spinning) spectra and spectral simulations of inhomogeneously broadened, stationary P-31 NMR lineshapes measured under CP (Cross-polarization) conditions. These methods revealed that the aminoacid Glu binds in the close realm of the phosphate in the hydrophilic headgroup of DPPC while β-Ala-Tyr is located more deeply inside the hydrophobic zone of the bilayer. The P-31 NMR simulations indicated restricted fast rotary motion of the phospholipids about their long axes in the organized bilayer structure. Finally, by the applied methodologies it is concluded that the two molecules under study exert dissimilar thermal and dynamic effects on lipid bilayers, the Glu improving significantly the packing of the lipids in contrast to the smaller and opposite effect of the dipeptide.     

Calorimetric study on the induction of interdigitated phase in hydrated DPPC bilayers by bioactive labdanes and correlation to their liposome stability: The role of chemical structure

Labd-7,13-dien-15-ol (1), labd-13-ene-8alpha,15-diol (2), and labd-14-ene-8,13-diol (sclareol) have been found to exhibit cytotoxic and cytostatic effects. Their partitioning into phospholipid bilayers may induce membrane structure modifications, crucial in the development of liposomes. DSC was used to elucidate the profile of modifications induced in DPPC bilayers by incorporating increasing concentrations of the labdanes. Labdanes 1, 2 and sclareol were incorporated into SUV liposomes composed of DPPC their physicochemical stability was monitored (4 degrees C) and was compared to liposomes incorporating cholesterol. All labdanes strongly affect the bilayer organization in a concentration dependent manner in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase, the induction of a lateral phase separation and the possible existence of interdigitated domains in the bilayer. The physicochemical stability of liposomes was strongly influenced by the chemical features of the labdanes. The liposomal preparations were found to retain their stability at low labdane concentration (10 mol%), while at higher concentrations up to 30 mol% a profound decrease in intact liposomes occurred, and a possible existence of interdigitated sheets was concluded.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

Direct measurement of phase coexistence in DPPC/cholesterol vesicles using Raman spectroscopy

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid-cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (l(d)), solid-ordered (s(o)) and liquid-ordered (l(o)) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at approximately 27 and approximately 22 mol% for l(d)-l(o) and s(o)-l(o) coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.