Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential scanning calorimetry and (2)H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane alpha-helical peptides on the organization of phosphatidylcholine bilayers

We have studied the effects of the incorporation of the alpha-helical transmembrane peptides Ac-K(2)-L(24)-K(2)-amide (L(24)) and Ac-K(2)-(L-A)(12)-K(2)-amide ((LA)(12)) on the thermotropic phase behavior of 1,2-dipalmitoyl-d(62)-sn-glycero-3-phosphocholine (DPPC-d(62)) and 1-palmitoyl-d(31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d(31)) lipid bilayer model membranes by differential scanning calorimetry (DSC) and the conformational and orientational order of the phospholipid chains by Fourier transform infrared (FTIR) spectroscopy and (2)H nuclear magnetic resonance ((2)H-NMR) spectroscopy, respectively. Our DSC and FTIR spectroscopic studies indicate that the peptides L(24) and (LA)(12) both decrease the temperature and enthalpy of the gel/liquid-crystalline phase transition of DPPC-d(62) bilayers, with (LA)(12) having the greater effect in this regard. An examination of the frequencies of the CH(2) and CD(2) symmetric stretching bands of the infrared spectra of liquid-crystalline states of the peptide-free and peptide-containing DPPC-d(62) and POPC-d(31) samples, and a comparison with the orientational order as measured by (2)H-NMR spectroscopy as well as with the chain order as measured by electron spin resonance spectroscopy, lead us to conclude that the CH(2) (or CD(2)) stretching frequencies of lipid hydrocarbon chains are not a reliable measure of chain conformational order in lipid bilayers containing significant amounts of peptides or other lipophilic inclusions. In contrast, the results of our (2)H-NMR spectroscopic studies present a consistent picture in which both L(24) and (LA)(12) increased in a similar way the time-averaged orientational order of the lipid chains of their liquid-crystalline lipid bilayer hosts. The comparison of the effects L(24) and (LA)(12) on phosphatidylcholine bilayers indicates that the gel-to-liquid-crystalline phase transition appears to be more sensitive to small changes in transmembrane peptide surface topology than hydrocarbon carbon chain orientational order in the liquid-crystalline state.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylcholine bilayers

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of an alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and odd-chain members of the homologous series of n-saturated diacylphosphatidylcholines. An analogue of L24, in which the lysine residues were all replaced by 2,3-diaminopropionic acid, and another, in which a leucine residue at each end of the polyLeu sequence was replaced by a tryptophan, were also studied. At low peptide concentrations, the DSC thermograms exhibited by these lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the transition temperature and cooperativity of this component, and its fractional contribution to the total enthalpy change, decrease with an increase in peptide concentration, more or less independently of phospholipid acyl chain length. The other component is very broad and predominates at high peptide concentrations. These two components have been assigned to the chain-melting phase transitions of populations of peptide-poor and peptide-enriched lipid domains, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that these peptides reduce somewhat but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the broad phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length. Our FTIR spectroscopic data indicate that these peptides form a very stable alpha-helix under all of our experimental conditions but that small distortions of their alpha-helical conformation are induced in response to mismatch between peptide hydrophobic length and gel-state bilayer hydrophobic thickness. We also present evidence that these distortions are localized to the N- and C-terminal regions of these peptides. Interestingly, replacing the terminal Lys residues of L24 by 2,3-diaminopropionic acid residues actually attenuates the hydrophobic mismatch effects of the peptide on the thermotropic phase behavior of the host PC bilayer, in contrast to the predictions of the snorkel hypothesis. We rationalize this attenuated hydrophobic mismatch effect by postulating that the 2,3-diaminopropionic acid residues are too short to engage in significant electrostatic and hydrogen-bonding interactions with the polar headgroups of the host phospholipid bilayer, even in the absence of any hydrophobic mismatch between incorporated peptide and the bilayer. Similarly, the reduced hydrophobic mismatch effect also observed when the two terminal Leu residues of L24 are replaced by Trp residues is rationalized by considering the lower energetic cost of exposing the Trp as opposed to the Leu residues to the aqueous phase in thin PC bilayers and the higher cost of inserting the Trp as opposed to the Leu residues into the hydrophobic cores of thick PC bilayers.     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and FTIR spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic model hydrophobic peptide, Lys2-Gly-Leu24-Lys2-Ala-amide, and members of the homologous series of n-saturated diacylphosphatidylcholines. In the low range of peptide mole fractions, the DSC thermograms exhibited by the lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the fractional contribution of this component to the total enthalpy change, the peak transition temperature, and cooperativity decrease with an increase in peptide concentration, more or less independently of acyl chain length. The other component is very broad and predominates in the high range of peptide concentration. These two components have been assigned to the chain-melting phase transitions of populations of bulk lipid and peptide-associated lipid, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that this peptide reduces but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length and that gel phase immiscibility occurs when the hydrophobic thickness of the bilayer greatly exceeds the hydrophobic length of the peptide. The FTIR spectroscopic data indicate that the peptide forms a very stable alpha-helix under all of our experimental conditions but that small distortions of its alpha-helical conformation are induced in response to any mismatch between peptide hydrophobic length and bilayer hydrophobic thickness. These results also indicate that the peptide alters the conformational disposition of the acyl chains in contact with it and that the resultant conformational changes in the lipid hydrocarbon chains tend to minimize the extent of mismatch of peptide hydrophobic length and bilayer hydrophobic thickness.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L24, in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP2-Leu24-DAP2-amide (L24DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K2-W-L22-W-K2-amide (WL22W)), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the PE bilayers is altered by these peptides in a hydrophobic mismatch-independent manner, in contrast to the hydrophobic mismatch-dependent manner observed previously with zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) bilayers. Moreover, all three peptides reduce the phase transition temperature to a greater extent in PE bilayers than in PC and PG bilayers, indicating a greater disruption of PE gel-phase bilayer organization. Moreover, the lysine-anchored L24 reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L24DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K2-W-L22-W-K2-amide) only slightly attenuates the effects of this peptide on the chain-melting phase transition of the host PE bilayers. All three peptides form very stable alpha-helices in PE bilayers, but small conformational changes occur in response to mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. These results suggest that the lysine snorkeling plays a significant role in the peptide-PE interactions and that cation-pi-interactions between lysine and tryptophan residues may modulate these interactions. Altogether, these results suggest that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer but also by the electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the polar headgroups of PE bilayers.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Effect of staphylococcal delta-lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier transform infrared spectroscopic, and X-ray diffraction studies

We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our 31P NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are studied, the isotropic component of the 31P NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase 31P NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic 31P NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D(2)O. However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.     

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.     

Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid-state NMR

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.     

Study of the interaction of an alpha-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic alpha-helical transmembrane peptide, acetyl-K(2)-L(24)-K(2)-amide (L(24)), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L(24) decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L(24) in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L(24):PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L(24). As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and (2)H NMR spectroscopy study of the interaction of the L(24) and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between (2)H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L(24) suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Thermotropic properties of saturated mixed acyl phosphatidylethanolamines

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

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.     

New structural model for mixed-chain phosphatidylcholine bilayers

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Geometry and intrinsic tilt of a tryptophan-anchored transmembrane alpha-helix determined by (2)H NMR

We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix.     

Molecular dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine membranes containing transmembrane alpha-helical peptides with alternating leucine and alanine residues

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide [(LA)(12)] on the molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes were investigated using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels, and the results were compared with our earlier, similar study of Ac-K(2)L(24)K(2)-amide (L(24)) [Subczynski, W. K., Lewis, R. N. A. H., McElhaney, R. N., Hodges, R. S., Hyde, J. S., and Kusumi, A. (1998) Biochemistry 37, 3156-3164]. At peptide-to-POPC ratios between 1/10 and 1/40, both methods (covering a time scale of 100 ps-10 micros) detect the presence of a single homogeneous membrane environment for both peptides, suggesting that these peptides are both well dispersed and that POPC is exchanging rapidly between the boundary and the bulk domains. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane, studied by saturation-recovery EPR, decreases by a factor of about 2 by including 10 mol % (LA)(12) whereas incorporating L(24) has practically no effect. (LA)(12) increases the alkyl chain order of POPC more than L(24). L(24) increases hydrophobicity (decreases the degree of water penetration into the hydrophobic region of the membrane) more than does (LA)(12). We ascribe the much stronger effects of (LA)(12) on membrane order and dynamics to the increased roughness of its hydrophobic surface and also to the increased motional freedom of its leucine side chains. In L(24), the leucine side chains are packed tightly, giving a smooth hydrophobic surface. In (LA)(12), they are separated by the small methyl groups of the alanine side chains, giving them additional motional freedom and the ability to protrude between the phospholipid hydrocarbon chains. The frequency of gauche-trans isomerization of hydrocarbon chains and concentration of vacant pockets (voids) in the lipid bilayer are thus reduced, which decreases oxygen transport. This explanation was confirmed by calculating the orientational order of leucine side chains in (LA)(12) and L(24) from molecular dynamics simulation studies.     

Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.     

The effect of plant hormone abscisic acid on model membranes - differential scanning calorimetry and planar bilayer membranes investigations

The effect of abscisic acid on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) and on phosphatidylethanolamines (natural (PE) and dipalmitoylphosphatidylethanolamine (DPPE)) bilayers was investigated by differential scanning calorimetry (DSC). Abscisic acid eliminates the pretransition of DPPC, causes a downward shift of its temperature of melting (T_m) and broadens the melting peak without changing the enthalpy of melting. In natural PE bilayers interacting with abscisic acid a small decrease in the enthalpy of melting almost without change of T_m was detected, whereas in synthetic DPPE abscisic acid caused a small shift of T_m and small broadening of the melting peak without changing the enthalpy of melting. Abscisic acid increases the conductance to Na⁺ or K⁺ by three orders of magnitude in planar lipid membranes formed from PE monolayers and by less than two orders of magnitude in membranes formed from PC monolayers.