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 (Lc) to lamellar gel (Lβ) phase transition and that the higher temperature transition involves the conversion of the Lβ phase to the lamellar liquid-crystalline (Lα) phase. Moreover, the FTIR spectroscopic signatures observed at temperatures below the onset of the Lc/Lβ phase transitions are consistent with the coexistence of structures akin to a TMCL-like Lc phase and the L β 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 β/Lα phase transition temperature. Poor miscibility of these two components is also suggested by the complexity of the DSC thermograms observed at the Lβ/Lα phase transitions of these mixtures and with the complex relationship between their Lβ/Lα 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 Lβ/Lα phase transition temperatures.Fil: Frías, María de los Ángeles. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benesch, Matthew G. K.. University of Alberta; CanadáFil: Lewis, Ruthven N. A. H.. University of Alberta; CanadáFil: McElhaney, Ronald N.. University of Alberta; Canad

Effect of Variations in the Structure of a Polyleucine-Based α-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 α-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP(2)-Leu(24)-DAP(2)-amide (L(24)DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K(2)-W-L(22)-W-K(2)-amide (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 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 L(24) reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L(24)DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K(2)-W-L(22)-W-K(2)-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 α-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-π-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

Calorimetric, X-Ray Diffraction, and Spectroscopic Studies of the Thermotropic Phase Behavior and Organization of Tetramyristoyl Cardiolipin Membranes

The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was studied by differential scanning calorimetry, x-ray diffraction, (31)P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (L(c)′) to gel (L(β)) phase transition and the higher temperature endotherm to a L(β) to lamellar liquid-crystalline (L(α)) phase transition. At the L(c)′/L(β) phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from ∼11 Å to ∼50 Å, and this value decreases slightly at the L(β)/L(α) phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 Å to 43.5 Å at the L(c)′/L(β) phase transition, consistent with the loss of the hydrocarbon chain tilt of ∼12°, and decreases to 37.8 Å at the L(β)/L(α) phase transition. The calculated cross-sectional areas of the TMCL molecules are ∼79 Å(2) and ∼83 Å(2) in the L(c)′ and L(β) phases, respectively, and we estimate a value of ∼100 Å(2) in the L(α) phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L(c)′ phase, TMCL molecules possess tilted all-trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L(β) phase the untilted, all-trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L(α) phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L(c)′ to the L(β) and then to the L(α) phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our (31)P-NMR results indicate that although the polar headgroup retains some mobility in the L(c)′ phase, its motion is much more restricted in the L(β) and especially in the L(α) phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes