Farnesol-DMPC phase behaviour: a (2)H-NMR study

Involved in a number of diverse metabolic and functional contexts, farnesol is a central component of the mevalonate pathway, post-translationally attaches to proteins, and affects a number of other membrane-associated events. Despite farnesol's biological implications, a detailed analysis of how farnesol affects the physical properties and phase behaviour of lipid membranes is lacking. As (2)H-NMR spectra are sensitive to molecular motions and acyl chain orientation, they can be used to measure the degree of molecular order present in the system. Also, since the (2)H-NMR spectra of fluid and gel phase lipids are very different, they are sensitive probes of membrane phase equilibrium and can be used to determine fluid-gel phase boundaries. In this study, dimyristoyl phosphatidylcholine-d(54) (DMPC-d(54)) bilayers containing varying concentrations of trans-trans farnesol (2.5-20.0 mol%) are investigated over a range of temperatures (8-30 degrees C). Analysis of these spectra has led to the construction of a farnesol-DMPC-d(54) temperature-composition plot. We show that increasing concentrations of farnesol induce a decrease in the fluid-gel phase transition temperature and promote fluid-gel coexistence. Interestingly, farnesol does not seem to affect the quadrupolar splittings (Delta v(Q)) in the fluid phase, i.e., the organization of farnesol within the bilayer and its interaction with phospholipids does not appreciably influence acyl chain order in the fluid phase.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (²H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T₁ relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length

FT-IR spectroscopy has been used to evaluate the acyl chain conformational ordering of DMPC, DMPE, DMPA (pH 6 and 12), DMPG (pH 1 and 7), and DPPC, DPPE, DPPA (pH 6). The frequencies of the symmetric and antisymmetric methylene stretching vibrations were determined as a function of temperature. In the liquid-crystalline phase the frequencies show a qualitative dependence on the amount of chain disorder. Quantitative data for trans-gauche isomerization were obtained from the integral intensities of the conformation sensitive methylene wagging absorptions at ca. 1368 cm^–1 (gtg and gtg sequences), 1356 cm ^–1 (double gauche) and 1342 cm^–1 (end gauche). The integral band intensities were converted to the number of gauche conformers per acyl chain using the calibration factors published by Senak et al. (1991). At 69°C the highest number of gauche conformers excluding contributions from single gauche conformers and jogs (gtttg) are found for PCs (DMPC: 2.6; DPPC: 2.4), followed by DMPG^– (2.0), phosphatidylethanolamines (DMPE: 1.4; DPPE: 2.0), protonated DMPG (1.5), and phosphatidic acids (DPPA^–: 1.7; DMPA^–: 1.4, DMPA^2–: 1.7). From ²H-NMR measurements of perdeuterated samples of DMPC, DMPA, DPPC, and DPPA the quadrupolar splittings _Qi and the order parameter S _CDi of the CD₂-segments close to the chain ends could be determined whereas splittings in the plateau region of the chains could not be resolved. The quadrupolar splittings are affected by trans-gauche isomerization, long axis rotation, and restricted wobbling motions of the acyl chains. In the simplest assumption, the order parameter S_CD can be expressed as a product of a segmental order parameter S and a lhain order parameter S . For comparison of the different lipids we used average order parameters S_CD, obtained by averaging over all values, and S determined from the total number of gauche conformers per chain by FT-IR-spectroscopy, to calculate an empirical average chain order parameter S. The combination of ²H-NMR and FT-IR results allows the estimation of the relative extent of chain wobbling for the different lipid molecules. S is lowest for PCs (S 0.475) while PEs (S 0.51) and PAs (S0.52) show less chain wobbling.Abbreviations FT-IR Fourier transform infrared - ²H-NMR deuterium nuclear magnetic resonance - DMPC(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylcholine - DMPE(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylethanolamine - DMPA(–d₅₄) (perdeuterated) dimyristoyl-phosphatidic acid - DMPG dimyristoyl-phosphatidylglycerol - DPPC(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylcholine - DPPE(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylethanolamine - DPPA(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidic acid - gtg gauche ^±-trans-gauche^± - gtg gauche^±-trans-gauche^± - dg double gauche - eg end gauche Correspondence to: A. Blume     

New monocationic methylpalladium(II) compounds with several bidentate nitrogen-donor ligands: synthesis, characterisation and reactivity with CO

Two series of methylpalladium(II) compounds with mono and bidentate nitrogen-donor ligands, namely [Pd(N-N)₂(CH₃)][X] (N-N=phen (1a), dm-phen (1b) (dm-phen=4,7-dimethyl-1,10-phenanthroline), tm-phen 1c (tm-phen=3,4,7,8-tetramethyl-1,10-phenanthroline); X=OTf, PF₆ ⁻) and [Pd(N-N)(L)(CH₃)][OTf] (N-N=phen and L=py (1ad) (py=pyridine), N-N=phen and L=2-Ph-py (1ae) (2-Ph-py=2-phenyl-pyridine), N-N=phen and L=BzQ (1af) (BzQ=7,8-benzoquinoline), N-N=tm-phen and L=BzQ (1cf)), have been synthesised and fully characterised both in solid state and in solution. The crystal structures of [Pd(phen)₂(CH₃)][PF₆] and [Pd(phen)(2-Ph-py)(CH₃)][OTf] show a square planar coordination geometry for palladium with the monodentate ligand (one phen molecule plays this role in 1a) bound to the metal with its plane almost perpendicular to the coordination plane. In both structures the PdN bond length trans to the methyl is remarkably affected by its trans influence. The behaviour in solution is characterised for the first series of compounds by a dynamic process which makes the two N-N ligands equivalent, as corroborated by the ¹⁵N NMR analysis: only one averaged signal is shown for all of the four nitrogen atoms. No fluxional process is present for the compounds of the second series, and three main crosspeaks are shown in the ¹⁵N-¹H HMQC spectra. In particular, the signal of the ¹⁵N trans to the methyl group has a typical chemical shift, which differs from those of two ¹⁵N trans to each other. Both series of complexes are reacted with carbon monoxide and the reaction products are studied by ¹H NMR spectroscopy and, when possible, by isolating the acyl derivatives. The products of this reaction are affected by the nature of the second molecule of N-ligand.     

Synthesis and structure of cadmium and zinc complexes of dehydroacetic acid

The cadmium and zinc complexes of Dehydroacetic Acid (DHA) Zn(DHA)₂(H₂O)₂ and Cd(DHA)₂(H₂O)₂ were synthesized and the derivatives Zn(ADH)₂(DMSO)₂ and Cd(ADH)₂(DMSO)₂ were prepared through substitution of the water ligands by DMSO. To characterize structural differences between the Cd and Zn complexes, a series of analyses were carried out: ¹H, ¹³C, and ¹¹³Cd NMR in solution and ¹³C and ¹¹³Cd NMR in the solid state, infra-red spectra, thermo gravimetric analysis (TGA), differential calorimetric analysis (DSC) and elemental analysis (CHNO). The X-ray crystal structures of the complexes Zn(DHA)₂(DMSO)₂ and Cd(DHA)₂(DMSO)₂ are also reported. The coordination around the metal atoms in the solid state is best described as distorted octahedra. The two chelating DHA ligands define an equatorial plane and the axial positions are occupied by two monodentate DMSO ligands coordinated by oxygen atoms, in the trans,trans,trans configuration. Significant differences were found between the Cd and Zn coordination spheres, with the latter forming relatively looser octahedral complexes.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T_m) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T_m to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T_m value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Synthesis, characterization and crystal structures of homo- and hetero-substituents containing trans six-coordinate tin(IV) complexes of H2tmtaa (5,14-dihydro-6,8,15,17-tetramethyldibenzo[b,i][1,4,8,11]tetraazacyclotetradecine), and the solid state NMR spectra study of Sn

The reaction of Sn(tmtaa)Cl₂ (H₂tmtaa=5,14-dihydro-6,8,15,17-tetramethyldi-benzo[b,i][1,4,8,11]tetraazacyclotetradecine) and ammonium thiocyanate or sodium azide under a mild condition resulted in trans six-coordinate tmtaa tin(IV) complexes, Sn(tmtaa)X₂ (X=NCS, 1; X=N₃, 2). However, the treatment of Sn(tmtaa)Cl₂ and sodium picrate produced Sn(tmtaa)(Cl)(OC₆H₂ (2,4,6-3NO₂)) (3). Only one chloro atom of Sn(tmtaa)Cl₂ was substituted because of low nucleophilicity of the 2,4,6-trinitrophenolic anion in 3. Furthermore, because of the steric hindrance between the 2,4,6-trinitrophenolic group and the tmtaa ligand, which has a non-planar, saddle-shaped conformation, two chloro atoms cannot be substituted by two 2,4,6-trinitrophenolic groups simultaneously. All complexes were characterized by IR spectra, UV spectra, mass spectra, NMR spectra and elemental analyses, as well as DSC measurements. X-ray crystal structures of 1 and 3 reveal that the complexes retain the characteristic saddle-shaped configuration of H₂tmtaa but have adopted the trans geometry. Solid state ¹¹⁹Sn NMR spectroscopy was used to study the bonding environment in the series of six-coordinate trans Sn(IV) tmtaa complexes. It can be found that the ¹¹⁹Sn chemical shifts of the Sn(IV) tmtaa complexes are almost not influenced by the substituents.     

Conformational order of the hydrocarbon chains in lipid bilayers. A raman spectroscopic study

Raman spectra of dihexadecylphosphatidic acid (DHPA) and of dimyristoylphosphatidylcholine (DMPC) and its longer chain homologues have been obtained as a function of temperature in order to study the conformational order of the hydrocarbon chains in lipid bilayers. The frequency of the longitudinal acoustical (LA) vibration band is evaluated in terms of the length of all-trans chain segments. In the ordered phase, the chains are found to be overwhelmingly in the all-trans conformation. In the fluid phase, definite all-trans segments occur predominantly, the length of which coincides with the extension of the order parameter plateau known from deuterium magnetic resonance (DMR). The frequency of the skeletal optical (SO) trans vibration band leads to the same result, if evaluated under the assumption of vibrational decoupling by gauche bands in the fluid phase, thus lending support to this assumption. The intensity of this band determined from the band area increases linearly with chain length in the ordered phase and is independent of chain length in the fluid phase. Evaluating the intensity for the length of all-trans segments, the same result for the chain conformation is obtained as derived from the frequencies, with the additional information that the length of the all-trans segments in the fluid phase does not vary with chain length.     

Synthesis, spectroscopical characterization and the biological activity in vitro of new platinum(II) complexes with imidazo[1,5-a]-1,3,5-triazine derivatives and dimethylsulfoxide

A series of platinum(II) complexes with 6,8-dimethylimidazo[1,5-a]-1,3,5-triazin-4(3H)-one (6,8-DiMe-4-O-IMT) (I) and 6,8-dimethyl-2-thioxo-2,3-dihydroimidazo[1,5-a]-1,3,5-triazin-4(1H)-one (6,8-DiMe-4-O-2-S-IMT) (II) of formula trans-[PtCl₂(dmso)(6,8-DiMe-4-O-IMT)] (1a) and trans-[PtCl₂(dmso)(6,8-DiMe-4-O-2-S-IMT)] (2a) have been prepared and characterized with ¹H, ¹³C, ¹⁵N, ¹⁹⁵Pt NMR and IR. Significant ¹⁵N NMR upfield coordination shifts (81-96 ppm) of N(7) atom indicate this nitrogen atom as a coordination site. The multinuclear NMR and IR spectra indicate the square planar geometry with N(7) bonded heterocycles, S-bonded dimethylsulfoxide and two trans chloride anions. The platinum(II) complexes were tested for their antiproliferative activity in vitro against the cells of four human cell lines: SW707 rectal adenocarcinoma, A549 non-small cell lung carcinoma, T47D breast cancer and HCV29T bladder cancer. The activity of (1a, 2a) was lower than that of cisplatin.     

Behaviour of a glycosphingolipid with unsaturated fatty acid in phosphatidylcholine bilayers

N-(Oleoyl)galactosylceramide with perdeuterated acyl chain was prepared by partial synthesis, and studied by wide line ²H-NMR in phospholipid liposomes. Spectra were obtained for low glycolipid concentrations in bilayers of dimyristoyl-, distearoyl-, and 1-palmitoyl-2-oleoylphosphatidylcholines. In an attempt to isolate the effects of glycosphingolipid fatty acid cis unsaturation on glycolipid behaviour in membranes, spectral findings related to the above species were compared to literature NMR data for pure 1-palmitoyl-2-oleoylphosphatidylcholine bilayers in which the oleoyl chain of the phospholipid had been deuterated, and to analogously deuterated glycerol based lipids in Acholeplasma laidlawii membranes. The results for N-(oleoyl-d₃₃)galactosylceramide proved to be qualitatively and quantitatively very similar to published data dealing with glycerol based lipids at comparable temperatures. In addition, the results were strikingly similar for glycolipids dispersed in saturated and unsaturated phospholipid host matrices. It would appear that the primary effects of cis 9,10 fatty acid unsaturation in glycosphingolipids (at low concentration in fluid phospholipid membranes) are the same as those of fatty acid cis unsaturation in glycerolipids. It further appears that the overall dynamic behaviour of N-(oleoyl)galactosylceramide in fluid phospholipid membranes is very similar to that of glycerolipids with comparable acyl chains.     

H NMR spectroscopy as a tool to determine accurate photoisomerization quantum yields of stilbene-like ligands coordinated to rhenium(I) polypyridyl complexes

In this work, the use of proton nuclear magnetic resonance, ¹H NMR, was fully described as a powerful tool to follow a photoreaction and to determine accurate quantum yields, so called true quantum yields (Φ_true), when a reactant and photoproduct absorption overlap. For this, Φ_true for the trans-cis photoisomerization process were determined for rhenium(I) polypyridyl complexes, fac-[Re(CO)₃(NN)(trans-L)]⁺ (NN = 1,10-phenanthroline, phen, or 4,7-diphenyl-1,10-phenanthroline, ph₂phen, and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy). The true values determined at 365 nm irradiation (e.g. Φ_NMR = 0.80 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺) were much higher than those determined by absorption spectral changes (Φ_UV-Vis = 0.39 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺). Φ_NMR are more accurate in these cases due to the distinct proton signals of trans and cis-isomers, which allow the actual determination of each component concentration under given irradiation time. Nevertheless when the photoproduct or reactant contribution at the probe wavelength is negligible, one can determine Φ_true by regular absorption spectral changes. For instance, Φ_313 nm for free ligand photoisomerization determined both by absorption and ¹H NMR variation are equal within the experimental error (bpe: Φ_UV-Vis = 0.27, Φ_NMR = 0.26; stpy: Φ_UV-Vis = 0.49, Φ_NMR = 0.49). Moreover, ¹H NMR data combined with electronic spectra allowed molar absorptivity determination of difficult to isolate cis-complexes.     

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii: A 2H-NMR study

We have investigated by ²H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of Acholeplasma laidlawii B. This is the only ²-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristics condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the ²H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.     

Probing the gel to liquid-crystalline phase transition and relevant conformation changes in liposomes by 13C magic-angle spinning NMR spectroscopy

A straightforward way to visualize gel to liquid-crystalline phase transition in phospholipid membranes is presented by using ¹³C magic-angle spinning NMR. The changes in the ¹³C isotropic chemical shifts with increasing temperature are shown to be a sensitive probe of the main thermotropic phase transition related to lipid hydrocarbon chain dynamics and relevant conformational changes. The average value of the energy difference between trans and gauche states in the central C4–11 fragment of the DMPC acyl chain was estimated to be 4.02 ± 0.2 kJ mol^− 1 in the liquid crystalline phase. The reported spectral features will be useful in ¹³C solid state NMR studies for direct monitoring of the effective lipid chain melting allowing rapid uniaxial rotation of membrane proteins in the biologically relevant liquid-crystalline phase.     

Synthesis, characterization, spectroscopic and electrochemical properties of trans,trans,trans-bis(triphenyl phosphine) bis(aroyl hydrazonato)ruthenium(II) complexes

Four ruthenium (II) complexes of general formula Ru(PPh₃)₂(L)₂ have been synthesized and characterized. The spectroscopic and cyclic voltammetric studies of these complexes are also reported. X-ray crystal structure determination of two of the complexes reveal that Ru(II) occupies trans,trans,trans-(t,t,t) N₂O₂P₂ centrosymmetric octahedral environments, with the ligand pair occupying the equatorial plane. ³¹P NMR confirms the presence of two trans-PPh₃ groups in all the complexes. The transformation of the complexes in dichloromethane solution is studied by spectrophotometry and ³¹P NMR spectroscopy.     

A Fourier-transform infrared spectroscopic study of the polymorphic phase behavior of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; a polymerizable lipid which forms novel microstructures

We have investigated the phase characteristics of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC₂₃PC), a phosphatidylcholine with diacetylenic groups in the acyl chains, and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC), using Fourier-transform infrared spectroscopy (FTIR). Previous studies on the phase behavior of DC₂₃PC in H₂O have shown that DC₂₃PC exhibits: (1) formation of cylindrical structures (‘tubules’) by cooling fluid phase multilamellar vesicles (MLVs) through T_m (43° C), and 2) metastability of small unilamellar vesicles (SUVs) in the liquid-crystalline state some 40° C below T_m, with subsequent formation of a gel phase comprised of multilamellar sheets at 2° C. The sheets form tubules when heated and cooled through T_m. FTIR results presented here indicate that as metastable SUVs are cooled toward the transition to bilayer sheets, spectroscopic changes occur before the calorimetric transition as measured by a reduction in the CH₂ symmetric stretch frequency and bandwidth. In spite of the vastly different morphologies, the sheet gel phase formed from SUVs is spectroscopically similar to the tubule gel phase. The C-H stretch region of DC₂₃PC gel phase shows bands at 2937 and 2810 cm⁻¹ not observed in the saturated analog of DC₂₃PC, which may be related to perturbations in the acyl chains introduced by the diacetylenic moiety. The narrow CH₂ scissoring mode at 1470 cm⁻¹ and the prominent CH₂ wagging progression indicate that DC₂₃PC gel phase was highly ordered acyl chains with extended regions of all-trans methylene segments. In addition, the 13 cm⁻¹ reduction in the C  O stretch frequency (1733–1720 cm⁻¹) during the induction of DC₂₃PC gel phase indicates that the interfacial region is dehydrated and rigid in the gel phase.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.