Tetramyristoyl Cardiolipin Research Articles

Mimicking the inner mitochondrial membrane with curved supported lipid bilayers: A neutron reflectometry study

Cardiolipin (CL) is a phospholipid with an unusual molecular structure exhibiting four acyl chains bound to a polar headgroup. CL is found in curved regions within biological membranes, such as the poles of cylindrically shaped bacteria and the cristae in mitochondria. Like bacterial cells, mitochondria are characterised by two cell membranes. CL is exclusively found in the inner membrane of mitochondria (IMM), and it is vital for the proper functioning of this organelle. Together with CL, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main lipid components of the IMM. Interestingly, most of the CL in human tissues exhibits C18 acyl chains and alterations of its molecular structure are related to severe diseases. Here we investigated supported lipid bilayers (SLBs) composed of PC, PE and CL as model systems mimicking the IMM. In our experiments we used tetra-oleyl cardiolipin (TOCL), which exhibits four C18 acyl chains and is therefore close to the CL normally present in the IMM, and tetra-myristoyl cardiolipin (TMCL), which exhibits considerably shorter acyl chains, i.e. C14. All samples were investigated both in the presence and in the absence of Ca2+ ions. The structure of the produced samples was characterised by neutron reflectometry (NR). Our data indicate that all samples with TMCL were organised as a regular SLB with a small impact of TMCL on the bilayer structure. On the other hand, at a TOCL concentration above 10% mol and upon injection of Ca2+, we observed a large structural rearrangement of the initially formed SLB compatible with the formation of curved bilayer regions that protrude towards the bulk solvent. To the best of our knowledge, this is the first example where specular NR and off-specular scattering revealed buckled SLBs. This experimental evidence indicates the crucial role of CL acyl chain composition in favouring the proper folding of the IMM.

Effects of curcumin in the interaction with cardiolipin-containg lipid monolayers and bilayers

Curcumin, a plant polyphenol extracted from the Chinese herb turmeric, has gained widespread attention in recent years because of its multifunctional properties as antioxidant, antinflammatory, antimicrobial, and anticancer agent. Effects of the molecule on mitochondrial membranes properties have also been evidenced. In this work, the interaction of curcumin with models of mitochondrial membranes composed of dimyristoylphosphatidylcholine (DMPC) or mixtures of DMPC and 4 mol% tetramyristoylcardiolipin (TMCL) has been investigated by using biophysical techniques. Spectrophotometry and fluorescence allowed to determine the association constant and the binding energy of curcumin with pure DMPC and mixed DMPC/TMCL aqueous bilayers. The molecular organization of pure DMPC and cardiolipin-containing Langmuir monolayers at the air-water interface were investigated and the morphology of the monolayers transferred into mica substrates were characterized through atomic force microscopy (AFM). It is found that curcumin associates at the polar/apolar interface of the lipid bilayers and the binding is favored in the presence of cardiolipin. At 2 mol%, curcumin is well miscible with lipid monolayers, particularly with mixed DMPC/TMCL ones, where compact terraces formation characterized by a reduction of the surface roughness is observed in the AFM topographic images. At 10 mol%, curcumin perturbs the stability of DMPC monolayers and morphologically are evident terraces surrounded by cur aggregates. In the presence of TMCL, very few curcumin aggregates and larger compact terraces are observed. The overall results indicate that cardiolipin augments the incorporation of curcumin in model membranes highlighting the mutual interplay cardiolipin-curcumin in mitochondrial membranes.

Interactions of Monovalent and Divalent Cations with Cardiolipin Monolayers.

Cardiolipin is a mitochondrial phospholipid with four alkyl chains and two phosphate moieties. Tetramyristoyl cardiolipin (TMCL, (14:0)4CL) monolayers at the air-water interface are characterized by compression isotherms, which show a liquid expanded/liquid condensed phase transition. The phase transition surface pressure πc depends on the composition of the aqueous solution. In a calculation, this is attributed to the electrostatic double layer, which is induced by the head groups of the model membrane, and competitive ion binding. The intrinsic binding constant is large for protons ( KH = 10 L/mol) and small for monovalent cations ( KM (Na+, K+, Cs+) = 10-3 L/mol). The different intrinsic binding constants explain the non-monotonic behavior of πc on increasing the salt concentration: raising the monovalent salt concentration increases πc by charging the TMCL monolayer until 0.1 mol/L, then screening effects dominate and decrease πc by reducing the electrostatic repulsion between lipid head groups. When at fixed 0.15 mol/L NaCl concentration, the concentration of divalent cations is increased, πc decreases. The intrinsic binding constants of divalent cations follow the sequence Sr2+ < Mg2+ < Mn2+ ≈ Zn2+ ≈ Ca2+ ( KD,Ca = 1.2 L/mol). The predictive power of the calculations was tested with different solutions.

Dipole Modifiers Regulate Lipid Lateral Heterogeneity in Model Membranes

In this study we report on experimental observations of giant unilamellar liposomes composed of ternary mixtures of cholesterol (Chol), phospholipids with relatively low Tmelt (DOPC, POPC, or DPoPC) and high Tmelt (sphingomyelin (SM), or tetramyristoyl cardiolipin (TMCL)) and their phase behaviors in the presence and absence of dipole modifiers. It was shown that the ratios of liposomes exhibiting noticeable phase separation decrease in the series POPC, DOPC, DPoPC regardless of any high-Tmelt lipid. Substitution of SM for TMCL led to increased lipid phase segregation. Taking into account the fact that the first and second cases corresponded to a reduction in the thickness of the lipid domains enriched in low- and high-Tmelt lipids, respectively, our findings indicate that the phase behavior depends on thickness mismatch between the ordered and disordered domains. The dipole modifiers, flavonoids and styrylpyridinium dyes, reduced the phase segregation of membranes composed of SM, Chol, and POPC (or DOPC). The other ternary lipid mixtures tested were not affected by the addition of dipole modifiers. It is suggested that dipole modifiers address the hydrophobic mismatch through fluidization of the ordered and disordered domains. The ability of a modifier to partition into the membrane and fluidize the domains was dictated by the hydrophobicity of modifier molecules, their geometric shape, and the packing density of domain-forming lipids. Phloretin, RH 421, and RH 237 proved the most potent among all the modifiers examined.

Psoralen loaded liposomal nanocarriers for improved skin penetration and efficacy of topical PUVA in psoriasis

Psoralen in combination with ultraviolet A radiation (PUVA) is an FDA recommended therapy for clinical application in the management of severe recalcitrant psoriasis. Psoralen acts by intercalation of DNA and upon exposure to UV-A, it forms monoadducts which in turn induce apoptosis. Poor skin deposition, weak percutaneous permeability of psoralen and adverse effects of severe burning, blisters, pigmentation associated with conventional topical psoralen vehicles hinders the therapeutic efficacy and safety of topical PUVA. The aim of the present study is to formulate psoralen loaded liposomal nanocarriers for enhanced skin penetration, safety and efficacy of topical PUVA in psoriasis. Two different liposomal compositions i.e., cationic liposomes composed of DC-Chol, cholesterol and anionic liposomes composed of egg lecithin, cholesterol, tetramyristoyl cardiolipin were prepared for the topical delivery of psoralen. Liposomal carriers were characterized with respect to size, zeta potential, entrapment efficiency, stability, in vitro drug release and in vivo studies. Both liposomes were prepared with particle size of nearly 100nm. Zeta potential and entrapment efficiency of cationic liposomes were +25.8mV, 75.12% and anionic liposomes were −28.5mV, 60.08% respectively. Liposomal dermal distribution demonstrated higher penetration of both liposomal carriers over solution. Similarly, skin permeation study indicated 5 fold increase in permeation of psoralen with liposomal carriers. Topical application of psoralen liposomal gels on imiquimod induced psoriatic plaque model reduced the symptoms of psoriasis and levels of key psoriatic cytokines such as tumor necrosis factor-α, IL-17 and IL-22. In conclusion, the developed liposomal carriers of psoralen were found to be promising and can find application for optimal safety and efficacy of topical PUVA in psoriasis.

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 Lβ/Lα phase transition temperatures exists. A recent DSC and X-ray diffraction study of DPPG/TMCL mixtures report similar results (Prossnigg et al., 2010).

Thermotropic Behavior of Cardiolipin and Dimyristoylphosphatidylcholine Bilayers in the Presence and Absence of Calcium

Cardiolipin is an unusual lipid in that it is comprised of two diacylglycerols linked by a glycerol bridge. It is in found primarily in membranes in which electron transport is coupled to phosphorylation. There it plays a critical role in effective functioning of the electron transport chain. The mechanism by which cardiolipin plays this role may be through effects on bilayer properties as well as through direct interactions with protein complexes. In this study we investigate the influence of increasing the composition of tetramyristoyl cardiolipin (TMCL) on the thermotropic behavior of dimyristoyl phosphatidylcholine (DMPC) bilayers using differential scanning calorimetry (DSC). DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Lipid dispersions of DMPC/TMCL were prepared in which TMCL constituted 0, 5,10, 20, 50 and 100 mole percent of the total lipid. In these studies the Tm increased from 25 oC to 40 oC as the TMCL composition increased. Ca++ has been shown to have a plethora of in vivo effects on biological membranes and to induce alterations in lipid phase behavior of model membranes. Therefore we further investigated the thermotropic behavior of DMPC/TMCL bilayers in the presence of Ca++. Above 20 mole percent TMCL a distinct phase separation was induced. The effect of Ca++ on cardiolipin/PC membranes was also investigated using 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN). These data are consistent with an alteration of the hydration of the bilayer in the headgroup region in the presence of Ca++ and suggest that Ca++ induces tighter packing.

Full picture of the thermotropic phase behavior of cardiolipin bilayer in water: identification of a metastable subgel phase

The formation of a stable crystalline phase from a fluid phase usually involves the passage through a gel or metastable subgel phase during cooling. Herein, we found for the first time that for the four-tailed lipid, tetramyristoyl cardiolipin (TMCL), a metastable subgel phase (“subgel II”-type phase, or SGII) was formed upon cooling from the gel phase, and such a metastable subgel phase served as the precursor towards the formation of a stable crystalline phase. A detailed thermotropic, X-ray diffraction and infrared spectroscopic study was carried out to study the formation of the subgel and crystalline phases, along with a comparison of the gel and fluid phases. All the four phases (crystalline, subgel, gel and fluid) are lamellar structured and the interlamellar spacings of the gel and fluid phases swell as the water content increases in pure water. A full picture of the thermotropic phase behavior of TMCL in water was obtained and it was found that the transformations between fluid–gel and gel–subgel are reversible, while the phase transformation processes involving the crystalline phase are irreversible. The gel to subgel transition mainly involves the reorganization of the tail packing state (from hexagonal to orthorhombic), and this phase transition was found to be independent of solvent. Changing from subgel to crystalline phase needs a long incubation time at relatively low temperatures (0–5 °C), and this crystalline phase converts to a gel phase upon heating. The present work sheds light on the crystallization process of amphiphiles and has profound implications on the stability of cardiolipin-based model systems or supramolecular self-assembly systems.

X-ray structure, thermodynamics, elastic properties and MD simulations of cardiolipin/dimyristoylphosphatidylcholine mixed membranes

Cardiolipins (CLs) are important biologically for their unique role in biomembranes that couple phosphorylation and electron transport like bacterial plasma membranes, chromatophores, chloroplasts and mitochondria. CLs are often tightly coupled to proteins involved in oxidative phosphorylation. The first step in understanding the interaction of CL with proteins is to obtain the pure CL structure, and the structure of mixtures of CL with other lipids. In this work we use a variety of techniques to characterize the fluid phase structure, material properties and thermodynamics of mixtures of dimyristoylphosphatidylcholine (DMPC) with tetramyristoylcardiolipin (TMCL), both with 14-carbon chains, at several mole percentages. X-ray diffuse scattering was used to determine structure, including bilayer thickness and area/lipid, the bending modulus, KC, and SXray, a measure of chain orientational order. Our results reveal that TMCL thickens DMPC bilayers at all mole percentages, with a total increase of ∼6Å in pure TMCL, and increases AL from 64Å2 (DMPC at 35°C) to 109Å2 (TMCL at 50°C). KC increases by ∼50%, indicating that TMCL stiffens DMPC membranes. TMCL also orders DMPC chains by a factor of ∼2 for pure TMCL. Coarse grain molecular dynamics simulations confirm the experimental thickening of 2Å for 20mol% TMCL and locate the TMCL headgroups near the glycerol-carbonyl region of DMPC; i.e., they are sequestered below the DMPC phosphocholine headgroup. Our results suggest that TMCL plays a role similar to cholesterol in that it thickens and stiffens DMPC membranes, orders chains, and is positioned under the umbrella of the PC headgroup. CL may be necessary for hydrophobic matching to inner mitochondrial membrane proteins. Differential scanning calorimetry, SXray and CGMD simulations all suggest that TMCL does not form domains within the DMPC bilayers. We also determined the gel phase structure of TMCL, which surprisingly displays diffuse X-ray scattering, like a fluid phase lipid. AL=40.8Å2 for the ½TMCL gel phase, smaller than the DMPC gel phase with AL=47.2Å2, but similar to AL of DLPE=41Å2, consistent with untilted chains in gel phase TMCL.

Simultaneous Detection of Nonpolar and Polar Compounds by Heat-Assisted Laser Ablation Electrospray Ionization Mass Spectrometry

A heat-assisted laser ablation electrospray ionization (HA-LAESI) method for the simultaneous mass spectrometric analysis of nonpolar and polar analytes was developed. The sample was introduced using mid-infrared laser ablation of a water-rich target. The ablated analytes were ionized with an electrospray plume, which was intercepted by a heated nitrogen gas jet that enhanced the ionization of analytes of low polarity. The feasibility of HA-LAESI was tested by analyzing, e.g., naphtho[2,3-a]pyrene, cholesterol, tricaprylin, 1,1',2,2'-tetramyristoyl cardiolipin, bradykinin fragment 1-8, and 1-palmitoyl-2-oleoyl-sn-glycerol. HA-LAESI was found better suited for low polarity compounds than conventional LAESI, whereas polar compounds were observed with both techniques. The sensitivity of HA-LAESI for the polar bradykinin fragment 1-8 was slightly lower than observed for LAESI. HA-LAESI showed a linear response for 500 nM to 1.0 mM solutions (n = 11) of verapamil with R(2) = 0.988. HA-LAESI was applied for the direct analysis of tissue samples, e.g., avocado (Persea americana) mesocarp and mouse brain tissue sections. Spectra of the avocado showed abundant triglyceride ion peaks, and the results for the mouse brain sections showed cholesterol as the main species. Conventional LAESI shows significantly lower ionization efficiency for these neutral lipids. HA-LAESI can be applied to the analysis of nonpolar and polar analytes, and it extends the capabilities of conventional LAESI to nonpolar and neutral compounds.

Membrane fluidity of tetramyristoyl cardiolipin (TMCL) liposomes studied by chronoamperometric monitoring of their adhesion and spreading at the surface of a mercury electrode

Giant unilamellar liposomes of the synthetic cardiolipin 1′,3′-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol give chronoamperometric current peaks at a stationary mercury electrode. The signals are due to the adhesion and spreading of the liposomes on the hydrophobic mercury surface. The potential dependence shows a minimum of the peak frequency at the point of zero charge, a large maximum of peak frequency at about −0.2 V and a second, however, smaller maximum at −0.8 V. The electrochemical behaviour of the liposomes indicates phase transitions of the cardiolipin which could be also observed in differential scanning calorimetry.

Hyaluronan synthase mediates dye translocation across liposomal membranes

BackgroundHyaluronan (HA) is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS), which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS) to liposomes preloaded with the fluorophore Cascade Blue (CB).ResultsCB translocation (efflux) was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL). An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL) and that, in contrast, tetramyristoyl cardiolipin (TM-CL) is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006). Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL.ConclusionsThe results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

Molecular Dynamics Simulation, 31P-NMR Spectroscopy, and Microelectrophoretic Studies of Cardiolipin Bilayers

Molecular dynamics (MD) simulations of tetramyristoyl cardiolipin (TMCL) and tetraoleoyl cardiolipin (TOCL) were carried out with the newly developed CHARMM C36 lipid force field (FF), and with head group charges q= −1 and −2. The surface areas per lipid, AL, for q=-2 are 127.1 ± 0.4 A2 for TOCL and 113.2 ± 0.2 A2 for TMCL. To validate the simulation results, 70% TOCL + 30% POPC bilayer was prepared both in simulation and NMR experiment. The order parameters of POPC from simulations were higher than those from experiment. Sodium-lipid van der Waals interactions were slightly reduced through CHARMM NBFIX. This change of parameters led to 5% increase in surface area/lipid, and substantially improved agreement with experiment for q = -2 TOCL. The net charge of cardiolipin at neutral pH has been the subject of debate. Microelectrophoresis and 31P-NMR spectroscopy experiments were carried out, and indicate that the net charge is −2.

Molecular Dynamics Simulation Studies of Cardiolipin Bilayers

Molecular dynamics (MD) simulations of tetramyristoyl cardiolipin (TMCL) and tetraoleoyl cardiolipin (TOCL) were carried out with the newly developed CHARMM lipid force field (FF), C36, and with head group charges q= −1 and −2. The surface areas per lipid, AL, for q=-1 are 126 ± 0.1 Å2 for TOCL and 111 ± .1 Å2 for TMCL. These are 1.8 times than those of the diacyl equivalents: 63 Å2 for dimyristoylphosphatidylcholine (DMPC) at 328K, and 69 Å2 for dioleoylphosphatidylcholine (DOPC) at 310K. Area compressibility, Ka, of TOCL equals 340 ± 40 dyn/cm, approximately 50% higher than experimentally obtained for DOPC (and most diacyl lipids); an experimental value for Ka for cardiolipins is not presently available. The areas and compressibilities for TOCL from the present simulation differ substantially from those obtained by Dahlberg and Maliniak using the FF of Berger et al. under the same conditions (AL= 99 Å2; Ka = 1100 dyn/cm). The origin of the differences appears to be in the ion binding to the surfaces of the cardiolipin bilayer. Under FF of Berger et al., ions bind closer to the carbonyl group in the lipid chain region whereas C36 CHARMM FF results ion binding closer to the negatively charged phosphate groups of the head group. Deuterium order parameter measurements are underway to determine which FF yields areas more representative of the fluid state of cardiolipin bilayers.

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 <80mol 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 20mol%. 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 Lβ phase, and with the relative amount of the TMCL-like Lc 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 Lβ/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.

Packing behaviour of two predominant anionic phospholipids of bacterial cytoplasmic membranes

Phosphatidylglycerol and cardiolipin represent the most abundant anionic phospholipid components of cytoplasmic bacterial membranes and thus are used as constituents for membrane mimetic systems. In this study, we have characterized the temperature dependent phase behaviour of the binary system dipalmitoyl-phosphatidylglycerol (DPPG) and tetramyristoyl-cardiolipin (TMCL) using microcalorimetry and X-ray scattering techniques. Both lipids exhibited a very similar main transition temperature (∼41°C), showing a minimum (39.4°C) for the binary mixtures at XDPPG=0.8, and exhibited low-temperature phase transitions, which were abolished by incorporation of small amounts (≤10mol%) of the other lipid component. Therefore, over a wide temperature and composition range a lamellar Lβ gel phase is the predominant structure below the chain melting transition, characterized by a relatively broad wide-angle peak for XDPPG≤0.8. This observation suggests the existence of packing inconsistencies of the TMCL/DPPG hydrocarbon lattices in the gel phase, supported by the small average size of lipid clusters (∼50 lipids) within this composition range. The bilayer thickness for the lamellar-gel phase showed a monotonic increase (56Å for TMCL to about 58Å for XDPPG=0.8 at 30°C), which may be explained by different degrees of partial interdigitation of the acyl chains to compensate for the differences in the hydrocarbon lengths of DPPG and TMCL in the Lβ phase.

Temperature Dependence of the Interaction of Antimicrobial Peptides With Mixed Lipid Bilayers

The interactions of two α-helical antimicrobial peptides, aurein 1.2 (13 residues) and maculatin 1.1 (21 residues), with model membranes have been examined using solid-state NMR and surface plasmon resonance techniques. P-31 NMR of multilamellar (MLV) dimyristoylphosphatidycholine (DMPC) vesicles with aurein 1.2 revealed minor disruptions in the bilayer above the gel-liquid phase transition. However, below the phase transition temperature an isotropic signal was observed, indicating that the peptide disrupted the bilayer and formed small, rapidly tumbling aggregates ∼ 22 nm in diameter as determined by light scattering measurements. However, the isotropic signal was not seen with the longer peptide. Additional experiments conducted using different lipid compositions revealed that both fluidity and temperature influence the peptide interaction. Gel phase lipid bilayers were more strongly affected by the peptide although similar effects were observed at lower temperatures in unsaturated chain lipid bilayers in the liquid crystalline state.A preliminary study on membranes mimicking the lipid composition of S. aureus has demonstrated a disruptive effect on the bilayer organization by addition of maculatin 1.1, a potent antibacterial peptide. As revealed in P-31 static NMR spectra of MLV composed of dimyristoylphosphatidylglycerol (DMPG) and tetramyristoylcardiolipin (TMCL), the peptide promoted formation of a dominant isotropic phase at 15°C, well below the liquid-crystalline transition temperature; while the lamellar organization was mainly restored above 50°C and an intermediate state was observed at 30°C. Interestingly, relaxation experiments on MLV without peptide indicated coexistence of two populations in the temperature range 30-50°C, most likely composed of fluid DMPG and rigid TMCL. The antimicrobial peptide may insert preferentially at domain boundaries, using defects in membrane packing to lower energy costs. Further experiments are ongoing to determine the nature of the isotropic phase and its relevance to antimicrobial activity.

On the Miscibility of Cardiolipin with the Major Lipid Components of the Bacterial Inner Membrane

The thermotropic phase behavior and organization of model membranes derived from binary mixtures of tetramyristoylcardiolipin (TMCL) with dimyristoyl phosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) were studied by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR) spectroscopy. Cardiolipin-containing DMPE and DMPG model membranes all exhibit complex multi-component hydrocarbon chain-melting (Lβ/Lα) phase transitions upon heating and cooling.

An Isocratic HPLC Method for the Simultaneous Determination of Cholesterol, Cardiolipin, and DOPC in Lyophilized Lipids and Liposomal Formulations

Phospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,1',2,2'-tetramyristoyl cardiolipin, along with cholesterol, form liposomes in aqueous media and have been investigated at NeoPharm (Lake Bluff, IL) as drug-delivery systems. To accurately assess the effectiveness of various formulations involving the use of aforementioned phospholipids and cholesterol, their quantitative determination is essential. An isocratic high-performance liquid chromatographic method for the simultaneous determination of cholesterol, cardiolipin, and DOPC in various pharmaceutical formulations containing the active drug substance has consequently been developed and is presented here. The current method utilizes an ASTEC-diol analytical column and is shown to be stability-indicating and free from interference from any of the formulation excipients, such as sucrose, sodium chloride, and sodium lactate. The analytes are detected using an evaporative light scattering detector (Alltech or Polymer Laboratories). The quantitation of each lipid component is performed using non-linear regression analysis. The retention characteristics of the analytes are examined as a function of eluent composition (e.g., pH, salt content, organic to aqueous phase ratio) and column temperature. The method was validated and was found to be sensitive, specific, rugged, and cost-effective. The current method provides enhanced chromatographic separation for lipid components as well as degradation products as compared to similar methods reported in the literature. It is also inherently simpler than other similar methods reported in the literature that typically use complex gradient elution.

Morphology modifications in negatively charged lipid monolayers upon mitochondrial creatine kinase binding

Mitochondrial creatine kinase (mtCK) may participate to membrane organization at the mitochondrial level by modulating lipid state and fluidity. The effect of the protein on lipid phase behaviour of different acyl chain length phosphatidylglycerol monolayers was analyzed from pressure-area isotherms and from the compressional modulus variation with respect to the surface pressure. Monolayer morphology was visualized by Brewster angle microscopy. No condensation effect was visible on dimyristoylphosphatidylglycerol (DMPG). For the other PG monolayers tested, dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG), mtCK facilitated the formation of a liquid condensed phase. The effect depended on the surface pressure at which transition phase occurred. The effect of mtCK was more pronounced for tetramyristoylcardiolipin (TMCL) monolayers, as liquid condensed regions appeared 10 mN/m below the transition phase of the pure TMCL monolayer. The observed domains were circular and rather uniform, indicating a stabilization of the condensed phase. The same effect, namely an overall condensation of the monolayer with formation of circular domains, was observed upon protein injection beneath TMCL monolayers in different condensation states at constant area. MtCK ability to induce and stabilize a LC phase on monolayers could have important consequences in membrane organization and emphasize its structural role at mitochondrial level.