A calorimetric and spectroscopic comparison of the effects of cholesterol and its sulfur-containing analogs thiocholesterol and cholesterol sulfate on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes.

We performed differential scanning calorimetric (DSC) and Fourier transform infrared (FTIR) spectroscopic studies of the effects of cholesterol (Chol), thiocholesterol (tChol) and cholesterol sulfate (CholS) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. Our DSC results indicate that Chol and tChol incorporation produce small temperature increases in the main phase transition broad component while CholS markedly decreases it, but Chol decreases cooperativity and enthalpy more strongly than CholS and especially tChol. Hence, Chol and tChol thermally stabilize fluid DPPC bilayer sterol-rich domains while CholS markedly destabilizes them, and CholS and particularly tChol are less miscible in such domains. Our FTIR spectroscopic results indicate that Chol incorporation increases the rotational conformational order of fluid DPPC bilayers to a slightly and somewhat greater degree than tChol and CholS, respectively, consistent with our DSC findings. Also, Chol and CholS produce comparable degrees of H-bonding (hydration) of the DPPC ester carbonyls in fluid bilayers, whereas tChol increases H-bonding. At low temperatures, Chol is fully soluble in gel-state DPPC bilayers, whereas tChol and CholS are not. Thus tChol and CholS incorporation can produce considerably different effects on DPPC bilayers. In particular, the tChol thiol group markedly reduces its lateral miscibility and increases DPPC carbonyl H-bonding without significantly affecting the other characteristic effects of Chol itself, while the CholS sulfate group significantly reduces its ability to thermally stabilize and order fluid DPPC membranes. This latter result suggests that the molecular basis for the purported ability of CholS to "stabilize" various biological membranes should be re-examined.

A calorimetric and spectroscopic comparison of the effects of cholesterol and its immediate biosynthetic precursors 7-dehydrocholesterol and desmosterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes.

We performed differential scanning calorimetric (DSC) and Fourier transform infrared (FTIR) spectroscopic studies of the effects of cholesterol (CHOL), 7-dehydrocholeterol (7DHC) and desmosterol (DES) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. 7DHC and DES are the immediate biosynthetic precursors of CHOL in the Kandutch-Russell and Bloch pathways and 7DHC and DES differ in structure from CHOL only by the presence of an additional double bond at C7 of ring B or C24 of the alkyl side chain, respectively. Our DSC results indicate that the incorporation of all three sterols produces comparable decreases in the temperature of the pretransition of DPPC, but CHOL decreases its cooperativity and enthalpy more strongly than 7DHC and especially DES. These findings indicate that all three sterols decrease the thermal stability of gel phase DPPC bilayers but that 7DHC and especially DES are less miscible in them. However, the incorporation of CHOL and DES produce comparable increases in the temperature of the broad component of the main phase transition of DPPC while 7DHC decreases it, but again CHOL produces greater decreases in its cooperativity and enthalpy then 7DHC and especially DES. These results indicate that CHOL and DES stabilize the sterol-rich domains of fluid DPPC bilayers, but that 7DHC and especially DES are less miscible in them. Our FTIR spectroscopic results indicate that CHOL increases the rotational conformational order of fluid DPPC bilayers to a somewhat and markedly greater degree than DES and 7DHC, respectively, consistent with our DSC findings. Our spectroscopic results also indicate that although all three sterols produce comparable degrees of H-bonding (hydration) of the DPPC ester carbonyl groups in fluid bilayers, CHOL is again found to be fully soluble in gel state DPPC bilayers at low temperatures, whereas 7DHC and especially DES are not. In general, we find that 7DHC and DES incorporation produce considerably different effects on DPPC bilayer membranes. In particular, the presence of an additional double bond at C7 or C24 produces a marked reduction in the ability of 7DHC to order fluid DPPC bilayers and in the miscibility of DES in such bilayers, respectively. These different effects may be the biophysical basis for the reduction of these double bonds in the last steps of CHOL biosynthesis, and for the deleterious biological effects of the accumulation of these sterols in vivo.

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: Binary mixtures of dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin.

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, nominally dianionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained, monoanionic phospholipid dimyristoylphosphatidylglycerol (DMPG) were examined by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The gel/liquid-crystalline phase transitions observed in these mixtures by DSC are generally rather broad and exhibit complex endotherms over a range of compositions. However, the phase transition temperatures and enthalpies exhibit nearly ideal behavior. Also, FTIR spectroscopic detection of the formation of stable and metastable DMPG-like lamellar crystalline (Lc) phases only at high DMPG levels upon low temperature annealing, and stable TMCL-like Lc phases at all higher TMCL concentrations, indicates that at low temperatures, laterally segregated domains of these two phospholipids must form, from which these different Lc phases nucleate and grow. Comparison of these results with those of a previous study of DMPE/TMCL mixtures (Frias et al., 2011) indicates that DMPG mixes slightly less well with TMCL than DMPE, perhaps because of the negative charge of the latter. However, in both binary mixtures, TMCL inhibits the formation of the Lc phase by DMPE even more strongly than for DMPG. Overall, our data suggest that TMCL and DMPG actually mix well across a broad temperature and composition range when the fatty acid chains of the two components are identical and only a modest (~17°C) difference between their Lß/Lα phase transition temperatures exists. A recent DSC and X-ray diffraction study of DPPG/TMCL mixtures report similar results (Prossnigg et al., 2010).

A comparative calorimetric and spectroscopic study of the effects of cholesterol and of the plant sterols ß-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 ß-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 DSC and FTIR spectroscopic study of the effects of the epimeric coprostan-3-ols and coprostan-3-one on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes: Comparison with their 5-cholesten analogues.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ß-OH, α-OH or CO) and in the presence or absence of a double bond in ring B and in the orientation of rings A and B. The Δ(5) sterols/steroid have a trans rather than a cis ring A/B junction, and the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases in the order ß-OH > α-OH > CO. However, in the saturated ring junction-inverted (cis) series, these concentrations are much more similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50 mol% sterol/steroid, which is inversely related to the miscibility of these compounds in fluid DPPC bilayers, also increases in the order ß-OH > α-OH > CO, but this effect is attenuated in the saturated series with an inverted ring A/B orientation. Moreover, replacement of the double bond at C5-C6 with a saturated linkage and inversion of the ring A/B junction reduces both sterol/steroid solubility and the ability to order the hydrocarbon chains of fluid DPPC molecules all cases. Thus, the characteristic effects of sterols/steroids on fluid lipid bilayers are generally optimal when an OH group rather than CO group is present at C3, and when this OH group is in the equatorial (ß) orientation, and when the orientation of the ring A/B fusion is trans rather than cis. Overall, these results demonstrate that variations in the saturation and stereochemistry of the steroid ring system influence the effect of variations in the nature and stereochemistry of the polar headgroup at C3 on the physical properties of phospholipid bilayers and vice versa. Moreover, the presence of a single double bond specifically at Δ(5) is required to maximize sterol solubility in fluid DPPC bilayers.

A DSC and FTIR spectroscopic study of the effects of the epimeric cholestan-3-ols and cholestan-3-one on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes: Comparison with their 5-cholesten analogs.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogs on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ß-OH, α-OH or C=O) and in the presence or absence of a double bond in ring B. In both the Δ(5) and saturated sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases in the order ß-OH > α-OH > C=O. However, in the saturated series, these concentrations are much more similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50 mol% sterol/steroid, inversely related to the miscibility of these compounds in fluid DPPC bilayers, also increases in the order ß-OH > α-OH > C=O, but this effect is again attenuated in the saturated series. Moreover, replacement of the double bond at C5 with a saturated linkage also reduces sterol/steroid solubility in all cases. Interestingly, the C5 double bond has no effect on DPPC hydrocarbon chain ordering in the ßOH sterol pair, considerably increases ordering in the αOH pair, and considerably reduces ordering in the C=O pair. Moreover, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ß-OH > α-OH > C=O in the Δ(5) series of compounds, but in the order ß-OH > C=O > α-OH in the saturated series. Our results indicate that the effects of the presence or absence of a double bond at C5 of ring B on the thermotropic phase behavior and organization of DPPC bilayers are influenced by the nature and stereochemistry of the polar group present at C3 and vice versa. Nevertheless, the characteristic effects of sterols/steroids on fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and when this OH group is in the equatorial (ß) orientation. Moreover, the presence of a single double bond specifically at C5 is required to maximize sterol solubility in fluid DPPC bilayers, which is probably its primary function in natural sterols.

A DSC and FTIR spectroscopic study of the effects of the epimeric 4,6-cholestadien-3-ols and 4,6-cholestadien-3-one on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ßOH, αOH or C=O) and in the position(s) of the double bond(s). In the Δ(5) sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases markedly in the order ßOH>αOH>C=O. However, in the Δ(4,6) series, these concentrations are similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50mol% sterol/steroid, which is inversely related to the miscibility of these compounds in the DPPC bilayer, also increases in the order ßOH>αOH>C=O, but this effect is attenuated in the Δ(4,6) series. In the two pairs of sterol epimers, the Δ(4,6) compounds exhibit a greater decrease in the temperature and enthalpy of both the pretransition and the main phase transition, whereas the opposite result is observed in the ketosteroid pair. Similarly, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ßOH>αOH>C=O in both series of compounds, but in the two pairs of sterol epimers, hydrocarbon chain ordering is greater for the Δ(5) than the Δ(4,6) sterols, whereas the opposite is the case for the steroid pair. Thus, the characteristic effects of sterols/steroids on fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and when this OH group is in the equatorial orientation. We suggest that the presence of keto-enol tautomerism in the conjugated Δ(4,6) ketosteroid may provide additional H-bonding opportunities to adjacent DPPC molecules in the bilayer, which results in more cholesterol-like effects.

Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes.

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely ß-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.

A DSC and FTIR spectroscopic study of the effects of the epimeric 4-cholesten-3-ols and 4-cholesten-3-one on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes: comparison with their 5-cholesten analogues.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five of its analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ßOH, αOH or C=O) and in the position of the double bond (C4-C5 in ring A or C5-C6 in ring B). In the three Δ(5) sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases in the order ßOH>αOH>C=O and these differences in concentration are significant. However, in the Δ(4) series, these concentrations are more similar, regardless of polar head group nature or stereochemistry. Similarly, the residual enthalpy of the main phase transition of DPPC at 50 mol.% sterol/steroid, which is inversely related to the miscibility of these compounds in the DPPC bilayer, also increases in the order ßOH>αOH>C=O, but this effect is attenuated in the Δ(4) as opposed to the Δ(5) series. Both of these results indicate that the presence of a double bond at C4-C5 in ring A, as compared to a C5-C6 double bond in ring B, reduces the effect of variations in the structure of the polar group at C3 on the properties of the host DPPC bilayer. The movement of the double bond from C5 to C4 in the two sterol pairs results in a greater decrease in the temperature and enthalpy of both the pretransition and the main phase transition, whereas the opposite result is observed in the ketosteroid pair. Similarly, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ßOH>αOH>C=O in both series of compounds, but in the two sterol pairs, hydrocarbon chain ordering is greater for the Δ(5) than the Δ(4) sterols, whereas the opposite is the case for the steroid pair. All of these results indicate that the typical effects of sterols/steroids in increasing the packing density and thermal stability of fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and that this OH group is more effective in the equatorial rather than the axial orientation. We can explain all of our sterol results by noting that the shift of the double bond from Δ(5) to Δ(4) introduces of a bend in ring A, which in turn destroys the coplanarity of the steroid fused ring system and reduces the goodness of sterol packing in the host DPPC bilayer. However, this conformational change should also occur in the ketosteroid pair, yet our experimental results indicate that the presence of the Δ(4) double bond is less disruptive than a double bond at Δ(5). We suggest that the presence of keto-enol tautomerism in the conjugated Δ(4) ketosteroid, but not in the nonconjugated Δ(5) compound, may provide additional H-bonding opportunities to adjacent DPPC molecules in the bilayer, which can overcome the unfavourable conformational change in ring A induced by the Δ(4) double bond.

Membrane lipid phase transitions and phase organization studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is a powerful yet relatively inexpensive and convenient technique for studying the structure and organization of membrane lipids in their various polymorphic phases. This spectroscopic technique yields information about the conformation and dynamics of all regions of the lipid molecule simultaneously without the necessity of introducing extrinsic probes. In this review, we summarize some relatively recent FTIR spectroscopic studies of the structure and organization primarily of fully hydrated phospholipids in their biologically relevant lamellar crystalline, gel and liquid-crystalline phases, and show that interconversions between these bilayer phases can be accurately monitored by this technique. We also briefly discuss how the structure and organization of potentially biologically relevant nonlamellar micellar or reversed hexagonal lipid phases can be studied and how phase transitions between lamellar and nonlamellar phases, or between various nonlamellar phases, can be followed as well. In addition, we discuss the potential for FTIR spectroscopy to yield fairly high resolution structural information about phospholipid packing in lamellar crystalline or gel phases. Finally, we show that many, but not all of these FTIR approaches can also yield valuable information about lipid-protein interactions in membrane protein- or peptide-containing lipid membrane bilayer model or even in biological membranes. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

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.

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: binary mixtures of dimyristoylphosphatidylethanolamine and tetramyristoylcardiolipin.

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, anionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained zwitterionic phospholipid dimyristoylphosphatidylethanolamine (DMPE) were examined by a combination of differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After equilibration at low temperature, DSC thermograms exhibited by binary mixtures of TMCL and DMPE containing < 80 mol DMPE exhibit a fairly energetic lower temperature endotherm and a highly energetic higher temperature endotherm. As the relative amount of TMCL in the mixture decreases, the temperature, enthalpy and cooperativity of the lower temperature endotherm also decreases and is not calorimetrically detectable when the TMCL content falls below 20 mol%. In contrast, the temperature of the higher temperature endotherm increases as the proportion of TMCL decreases, but the enthalpy and cooperativity both decrease and the transition endotherms become multimodal. The FTIR spectroscopic results indicate that the lower temperature endotherm corresponds to a lamellar crystalline (L(c)) to lamellar gel (L(ß)) phase transition and that the higher temperature transition involves the conversion of the L(ß) phase to the lamellar liquid-crystalline (L(α)) phase. Moreover, the FTIR spectroscopic signatures observed at temperatures below the onset of the L(c)/L(ß) phase transitions are consistent with the coexistence of structures akin to a TMCL-like L(c) phase and the L(ß) phase, and with the relative amount of the TMCL-like L(c) phase increasing progressively as the TMCL content of the mixture increases. These latter observations suggest that the TMCL and DMPE components of these mixtures are poorly miscible at temperatures below the L(ß)/L(α) phase transition temperature. Poor miscibility of these two components is also suggested by the complexity of the DSC thermograms observed at the L(ß)/L(α) phase transitions of these mixtures and with the complex relationship between their L(ß)/L(α) phase transition temperatures and the composition of the mixture. Overall, our data suggests that TMCL and DMPE may be intrinsically poorly miscible across a broad composition range, notwithstanding the homogeneity of the fatty acid chains of the two components and the modest (~10 °C) difference between their L(ß)/L(α) phase transition temperatures.

The effect of variations in phospholipid and sterol structure on the nature of lipid-sterol interactions in lipid bilayer model membranes.

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.

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.

The physicochemical properties of cardiolipin bilayers and cardiolipin-containing lipid membranes.

In this review article, we summarize the current state of biophysical knowledge concerning the phase behavior and organization of cardiolipin (CL) and CL-containing phospholipid bilayer model membranes. We first briefly consider the occurrence and distribution of CL in biological membranes and its probable biological functions therein. We next consider the unique chemical structure of the CL molecule and how this structure may determine its distinctive physical properties. We then consider in some detail the thermotropic phase behavior and organization of CL and CL-containing lipid model membranes as revealed by a variety of biophysical techniques. We also attempt to relate the chemical properties of CL to its function in the biological membranes in which it occurs. Finally, we point out the requirement for additional biophysical studies of both lipid model and biological membranes in order to increase our currently limited understanding of the relationship between CL structure and physical properties and CL function in biological membranes.

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.

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.

Differential scanning calorimetry in the study of lipid phase transitions in model and biological membranes: practical considerations.

Differential scanning calorimetry (DSC) is a relatively rapid, straightforward, and nonperturbing technique for studying the thermotropic phase behavior of hydrated lipid dispersions and of reconstituted lipid model or biological membranes. However, because of the diversity of lipid thermotropic phase behavior, data-acquisition and data-analysis protocols must be modified according to the nature of the phase transition under investigation. In this chapter, the theoretical basis of the DSC experiment is examined and, with the aid of specific examples, also how the information content of DSC thermograms is affected by the nature of the lipid phase transition examined. The overall goal is to provide practical guidelines for the development of data-acquisition and data-analysis protocols, which are compatible with the instrumentation available and the nature of the lipid phase transition under investigation.

Fourier transform infrared spectroscopy in the study of lipid phase transitions in model and biological membranes: practical considerations.

Fourier transform infrared (FTIR) spectroscopy is a powerful, nonperturbing technique that has been used to good effect for the detection and characterization of lipid phase transitions in model and natural membranes. The technique is also quite versatile, covering a wide range of sophisticated applications, from which fairly detailed information about the structure and organization of membranes and other lipid assemblies can be obtained. In this chapter, an introduction to this particular application of FTIR spectroscopy is presented. Special emphasis is put on how the technique can be used to study lipid phase transitions under biologically relevant conditions. The chapter is intended to give an overview of the capabilities of FTIR spectroscopy in the field of lipid and biomembrane research, and provide the reader with some practical guidelines for the design and execution of simple FTIR spectroscopic experiments suitable for the detection and characterization of lipid phase transitions in hydrated lipid bilayers.

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.