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.     

A comparative calorimetric and spectroscopic study of the effects of cholesterol and of the plant sterols β-sitosterol and stigmasterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of β-sitosterol (Sito) and stigmasterol (Stig) on the thermotropic phase behavior and organization of DPPC bilayers. Sito and Stig are the major sterols in the biological membranes of higher plants, whereas cholesterol (Chol) is the major sterol in mammalian membranes. Sito differs in structure from Chol in having an ethyl group at C24 of the alkyl side-chain, and Stig in having both the C24 ethyl group and trans-double bond at C22. Our DSC studies indicate that the progressive incorporation of Sito and Stig decrease the temperature and cooperativity of the pretransition of DPPC to a slightly lesser and greater extent than Chol, respectively, but the pretransition persists to 10 mol % sterol concentration in all cases. All three sterols produce essentially identical effects on the thermodynamic parameters of the sharp component of the DPPC main phase transition. However, the ability to increase the temperature and decrease the cooperativity and enthalpy of the broad component decreases in the order Chol > Sito > Stig. Nevertheless, at higher Sito/Stig concentrations, there is no evidence of sterol crystallites. Our FTIR spectroscopic studies demonstrate that Sito and especially Stig incorporation produces a smaller ordering of the hydrocarbon chains of fluid DPPC bilayers than does Chol. In general, the presence of a C24 ethyl group in the alkyl side-chain reduces the characteristic effects of Chol on the thermotropic phase behavior and organization of DPPC bilayer membranes, and a trans-double bond at C22 magnifies this effect.     

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.     

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)     

Comparative calorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (~30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.     

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.     

Sterol chemical configuration influences the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayers containing 5α-cholestan-3β- and 3α-ol

It is commonly believed that all membrane sterols are rigid all-trans ring systems with a fully extended alkyl side-chain and that they similarly influence phospholipid bilayer physical properties. Here, we report the sterol concentration-dependent, thermotropic phase behaviour of binary dipalmitoylphosphatidylcholine (DPPC)/sterol mixtures containing two similar 5α-H sterols with different functional group orientations (3α-OH or 3β-OH), which adopt an ideal all-trans planar ring conformation but lack the deformed ring B conformation of cholesterol (Chol) and epicholesterol (Echol), using differential scanning calorimetry (DSC). Our deconvolution of the DSC main phase transition endotherms show differences in the proportions of sterol-poor (sharp) and sterol-rich (broad) domains in the DPPC bilayer with increasing sterol concentration, which delineate gel/liquid-crystalline (P_β′/L_α) and disordered gel (L_β)/liquid-ordered (l_o) phase regions. There are similarities in the DPPC main phase transition temperature, cooperativity and enthalpy for each 3β-ol and 3α-ol pair with increasing sterol concentration and differences in the parameters obtained for both the sterol-poor and sterol-rich regions. The sterol-poor domain persists over a greater concentration range in both 3α-ol/DPPC mixtures, suggesting that either those domains are more stable in the 3α-ols or that those sterols are less miscible in the sterol-rich domain. Corresponding parameters for the sterol-rich domain show that at sterol concentrations up to 20 mol%, the 5α-H,3β-ol is more effective at reducing the phase transition enthalpy of the broad component () than Chol, but is less effective at higher concentrations. Although mixtures containing Echol and 5α-cholestan-3α-ol have similar positive slopes below 7 mol% sterol, suggesting that they abolish the L_β/l_o phase transition equally effectively at low concentrations, Echol is more effective than the saturated 3α-ol at higher sterol concentrations. A comparison of obtained for the saturated and unsaturated pairs suggests that the latter sterols stabilize the l_o phase and broaden and abolish the DPPC main phase transition more effectively than the saturated sterols at physiologically relevant concentrations, supporting the idea that the double bond of Chol and Echol promotes greater sterol miscibility and the formation of l_o phase lipid bilayers relative to corresponding saturated sterols in biological membranes.     

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.     

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.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Characterization of liposomal tacrolimus in lung surfactant-like phospholipids and evaluation of its immunosuppressive activity

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations 相似文献   

Biophysical perturbations induced by ethylazinphos in lipid membranes

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.     

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.     

Studies on the purified Na+,Mg(2+)-ATPase from Acholeplasma laidlawii B membranes: a differential scanning calorimetric study of the protein-phospholipid interactions

The purified Na+,Mg2(+)-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid-crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid-crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

A molecular basis explanation of the dynamic and thermal effects of vinblastine sulfate upon dipalmitoylphosphatidylcholine bilayer membranes

Differential scanning calorimetry has been employed to study the thermal effects of vinblastine sulfate upon aqueous, single and multiple bilayer dispersions of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC). The calorimetric results summarized to an increase in the gel to liquid-crystalline phase transition enthalpy and the abolishment of the L(beta)' (gel phase) to P(beta)' (ripple phase) pretransition for the uni- and multilamellar dispersions, as well as an increase in the transition temperature T(m) and the transition cooperativity for single bilayer DPPC/vinblastine mixed vesicles, are consistent with an induced, partially interdigitated, gel phase. Computational analysis has been successfully applied to clarify the intermolecular effects and verify the feasibility of the proposed interdigitation for the vinblastine sulfate molecules and also for the ursodeoxycholic acid (UDCAH) and bromocylated taxanes, which have been shown to induce an interdigitated gel phase in DPPC bilayers.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Molecular characteristics of astaxanthin and beta-carotene in the phospholipid monolayer and their distributions in the phospholipid bilayer.

The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.     

A calorimetric study of binary mixtures of dihydrosphingomyelin and sterols,sphingomyelin, or phosphatidylcholine

The thermotropic properties of binary mixtures of D-erythro-n-palmitoyl-dihydrosphingomyelin (16:0-DHSM), D-erythro-n-palmitoyl-sphingomyelin (16:0-SM), cholesterol, lathosterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by differential scanning calorimetry. Addition of sterol to 16:0-DHSM and 16:0-SM bilayers resulted in a progressive decrease in both the T(m) and the enthalpy of the main transition. The sterol-induced broad components in 16:0-DHSM endotherms had markedly lower enthalpies than those induced in 16:0-SM. Pretransitions recorded in 16:0-DHSM and 16:0-SM membranes responded differently to low concentrations of cholesterol. The presence of 5 mol % cholesterol increased the pretransition temperature in 16:0-SM bilayers, whereas it decreased the temperature in 16:0-DHSM membranes. Lathosterol behaved in general as cholesterol with regard to its effects on the thermotropic behavior of both sphingolipids, but it appeared to form more stable sterol-rich domains, as seen from the higher T(m) of the broad component, in comparison to cholesterol. Thermograms recorded on binary mixtures of 16:0-SM:16:0-DHSM and DPPC:16:0-DHSM showed that 16:0-SM mixed nearly ideally with 16:0-DHSM, whereas DPPC mixing was less ideal in a 16:0-DHSM membrane. In conclusion, we observed that 16:0-DHSM interactions with sterols differed from that seen with 16:0-SM, and that 16:0-DHSM mixed better with 16:0-SM than DPPC, which indicates that DHSM could function as a membrane organizer within laterally condensed domains.