Farnesol-DMPC phase behaviour: a 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 ²H-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 ²H-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-d₅₄ (DMPC-d₅₄) bilayers containing varying concentrations of trans-trans farnesol (2.5-20.0 mol%) are investigated over a range of temperatures (8-30 °C). Analysis of these spectra has led to the construction of a farnesol-DMPC-d₅₄ 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 (Δv_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.     

Lipid lateral segregation driven by diacyl cyclodextrin interactions at the membrane surface

Cyclodextrins are hydrophilic molecular cages with a hydrophobic interior allowing the inclusion of water-insoluble drugs. Amphiphilic cyclodextrins obtained by appending a hydrophobic anchor were designed to improve the cell targeting of the drug-containing cavities through their liposome transportation in the organism. After insertion in model membranes, they were found to induce a lateral phase separation into a pure lipid phase and a fluid cyclodextrin-rich phase (L(CD)) with reduced acyl chain order parameters, as observed with a derivative containing a cholesterol anchor (M. Roux, R. Auzely-Velty, F. Djeda?ni-Pilard, and B. Perly. 2002. Biophysical Journal, 8:813-822). We present another class of amphiphilic cyclodextrins obtained by grafting aspartic acid esterified by two lauryl chains on the oligosaccharide core via a succinyl spacer. The obtained dilauryl-beta-cyclodextrin (betaDLC) was inserted in chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) membranes and studied by deuterium NMR ((2)H-NMR). A laterally segregated mixed phase was found to sequester three times more lipids than the cholesteryl derivative (approximately 4-5 lipids per monomer of betaDLC), and a quasipure L(CD) phase could be obtained with a 20% molar concentration of betaDLC. When cooled below the main fluid-to-gel transition of DMPC-d54 the betaDLC-rich phase stays fluid, coexisting with pure lipid in the gel state, and exhibits a sharp transition to a gel phase with frozen DMPC acyl chains at 12.5 degrees C. No lateral phase separation was observed with partially or fully methylated betaDLC, confirming that the stability of the segregated L(CD) phase was governed through hydrogen-bond-mediated intermolecular interactions between cyclodextrin headgroups at the membrane surface. As opposed to native betaDLC, the methylated derivatives were found to strongly increase the orientational order of DMPC acyl chains as the temperature reaches the membrane fluid-to-gel transition. The results are discussed in relation to the "anomalous swelling" of saturated phosphatidylcholine multilamellar membranes known to occur in the vicinity of the main fluid-to-gel transition.     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

Deuterium magnetic resonance study of phase equilibria and membrane thickness in binary phospholipid mixed bilayers.

The gel-fluid phase equilibrium in a two-component system formed from dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) was investigated using solid-state wide-line 2H NMR spectroscopy. Analysis of the spectral first moments and the quantitation of gel and fluid phases by means of difference spectroscopy provided the temperature-composition phase diagrams. Phase diagrams were constructed for mixtures of perdeuterated DMPC, DMPC-d54, with DSPC and for the complementary system comprised of DMPC and perdeuterated DSPC, DSPC-d70. The gel-fluid coexistence region was found to extend over a wider range of temperature and composition for the DMPC-d54-DSPC system than for the DMPC-DSPC-d70 system. Comparison of these data with the phase diagram for the DMPC-DSPC system showed that in the gel-fluid region the fraction of lipids in the fluid phase at a given temperature and system composition decreases for the three systems in the order DMPC-d54-DSPC greater than DMPC-DSPC greater than DMPC-DSPC-d70. While the fluid fraction varies by as much as 90% among the three systems, the composition of the fluid phase, i.e., the ratio of the concentrations of the two molecules in the fluid phase, varies by about 20% over the whole temperature and system composition range. The effective acyl chain lengths of the DMPC-d54 and DSPC-d70 molecules as a function of temperature and composition in the fluid phase, when the system is all fluid or is in the gel-fluid coexistence region, were calculated from the quadrupole splittings in the axially symmetric powder patterns obtained for the all-fluid phase. The magnitudes of the coefficient of thermal expansion for both the DMPC-d54 and the DSPC-d70 molecules were smaller in the fluid phase of binary mixtures than in one-component bilayers containing either DSPC-d70 or DMPC-d54 alone. In addition, at any given temperature in the fluid phase, the increase in the acyl chain length of DMPC-d54 with increasing DSPC content of the system was smaller than the concomitant increase in the length of DSPC-d70 in mixtures with DMPC. In the entire temperature and composition range when the binary mixtures are in the all-fluid or in the gel-fluid coexistence region, the largest value obtained for the DMPC-d54 molecule in the fluid phase was smaller than the smallest value obtained for the DSPC-d70 molecule in the fluid phase. The acyl chain lengths were used to calculate the effective weighted-average thickness, d, of the fluid phase bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Phospholipid/cholesterol membranes containing n-alkanols: a 2H-NMR study

The influences of 1-octanol and 1-decanol on aqueous multilamellar dispersions of 1-hexadecanoyl(octadecanoyl)-2-[2H31]hexadecanoyl-sn-glycero -3-phosphorylcholine (PC-d31)/cholesterol (3:1) have been examined using 2H-NMR. The gel to liquid crystalline phase transition of the PC-d31/cholesterol dispersion is modulated by the addition of 1-alkanol, which reduces the onset temperature and increases the width of the transition. 1-Octanol has a greater effect on the transition onset and completion temperatures than does 1-decanol, as determined from analysis of the temperature-dependent 2H-NMR spectra. 2H-NMR C-2H bond order parameters as a function of phospholipid acyl chain position at 60 degrees C, where all dispersions are fully liquid crystalline, have been calculated from the depaked spectra. 1-Decanol reduces the phospholipid order by only 2%. This can be attributed to the lower effective cholesterol concentration in the 1-alkanol/PC-d31/cholesterol dispersions. 1-Octanol, however, reduces the phospholipid order by 10% at 60 degrees C. Correlations between the effects of 1-octanol and 1-decanol on phospholipid order parameters and phospholipid/cholesterol phase transitions are discussed.     

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.     

Dynamics and ordering of lipid spin-labels along the coexistence curve of two membrane phases: an ESR study

An analysis of electron spin resonance (ESR) spectra from compositions along the liquid-ordered (L(o)) and liquid-disordered (L(d)) coexistence curve from the brain-sphingomyelin/dioleoylphosphatidylcholine/cholesterol (SPM/DOPC/Chol) model lipid system was performed to characterize the dynamic structure on a molecular level of these coexisting phases. We obtained 200 continuous-wave ESR spectra from glycerophospholipid spin-labels labeled at the 5, 7, 10, 12, 14, and 16 carbon positions of the 2nd acyl chain, a sphingomyelin spin-label labeled at the 14 carbon position of the amide-linked acyl chain, a headgroup-labeled glycerophospholipid, a headgroup-labeled sphingomyelin, and the cholesterol analogue spin-label cholestane all within multi-lamellar vesicle suspensions at room temperature. The spectra were analyzed using the MOMD (microscopic-order macroscopic-disorder) model to provide the rotational diffusion rates and order parameters which characterize the local molecular dynamics in these phases. The analysis also incorporated the known critical point and invariant points of the neighboring three-phase triangle along the coexistence curve. The variation in the molecular dynamic structures of coexisting L(o) and L(d) compositions as one moves toward the critical point is discussed. Based on these results, a molecular model of the L(o) phase is proposed incorporating the "condensing effect" of cholesterol on the phospholipid acyl chain dynamics and ordering and the “umbrella model” of the phospholipid headgroup dynamics and ordering.     

The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR

By varying the pH, the influence of the ionization degree on the structure and dynamics of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was studied, using 2H-NMR methods. For this purpose DOPS was synthesized with deuterium labels incorporated either stereospecifically at the beta-position of the serine headgroup ([2-2H]DOPS) or at the 11-position of both acyl chains ([11,11-2H2]DOPS), allowing the effects of pH on headgroup and acyl chains to be measured in parallel. A large scale synthesis procedure of stereospecific 1,2-dioleoyl-sn-glycero-3-phospho-[2-2H]-L- serine is described. The quadrupolar splitting (delta nu q) of [2-2H]DOPS is shown to be a sensitive sensor for the degree of protonation of the molecule. Whereas the delta nu q of [2-2H]DOPS decreases upon lowering the pH, that of [11,11-2H2]DOPS gradually increases, indicating an increase in acyl chain ordering. In the pH range below the pKa value, DOPS exhibits a temperature-dependent bilayer to hexagonal HII phase transition, apparent from the 31P-NMR spectra and the occurrence of a second component in the [11,11-2H2]DOPS 2H-NMR spectrum, with a much smaller delta nu q. The HII phase component in spectra from [2-2H]DOPS coincides with the isotropic position and has no defined delta nu q. In the bilayer organization delta nu q and spin-lattice relaxation time (T1) values for the acyl chain deuterated DOPS are similar to those obtained for other lipid systems. In contrast the PS headgroup region displays a relatively rigid structure as evidenced by a large delta nu q and very small T1 values. Upon adopting the HII phase the T1 values of the acyl chain deuterons are hardly affected. The uniqueness of the PS headgroup with respect to structure and motional properties is reinforced by the occurrence of a T1 minimum at 45 degrees C in the measurement of the temperature dependence of T1 for [2-2H]DOPS in the hexagonal HII configuration. Quantitative analysis yields a correlation time (tau c) for the motions determining T1 under these conditions, of 3.45 ns.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of farnesol on the physical properties of DMPC membranes

Farnesol interacts with membranes in a wide variety of biological contexts, yet our understanding of how it affects lipid bilayers is not yet complete. This study investigates how the 15-carbon isoprenoid, farnesol, influences the phase behaviour, lateral organization, and mechanical stability of dimyristol phosphatidylcholine (DMPC) model membranes. Differential scanning calorimetry (DSC) of multilamellar DMPC-farnesol mixtures (up to 26 mol% farnesol) demonstrates how this isoprenoid lowers and broadens the gel-fluid phase transition. A gel-fluid coexistence region becomes progressively more dominant with increasing farnesol concentration and at concentrations of and greater than 10.8 mol%, an upper transition emerges at about 35 degrees C. Atomic force microscopy images of supported farnesol-DMPC bilayers containing 10 and 20 mol% farnesol provide structural evidence of gel-fluid coexistence around the main transition. Above this coexistence region, membranes exhibit homogeneous lateral organization but at temperatures below the main gel-fluid coexistence region, another form of phase coexistence is observed. The solid nature of the gel phase is confirmed using micropipette aspiration. The combined thermodynamic, structural, and mechanical data allow us to construct a phase diagram. Our results show that farnesol preferentially partitions into the fluid phase and induces phase coexistence in membranes below the main transition of the pure lipid.     

Elastic Interactions of Photosynthetic Reaction Center Proteins Affecting Phase Transitions and Protein Distributions

Reaction-center proteins of Rhodopseudomonas Sphaeroides reconstituted into phosphatidylcholine vesicles shift and broaden the fluid-gel transition of the lipid bilayer. The amount of broadening and temperature shift of the transition depend both on protein concentration and on lipid chain length. In particular, the direction of the transition temperature shift is very sensitive to lipid chain length. Electron micrographs show homogeneous protein distribution on the fluid surface whereas the solid phase contains protein aggregates the type depending on chain length. The results can qualitatively be understood in the framework of a mattress model of lipid/protein interactions in membranes.     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.     

Structure and lipid interaction of N-palmitoylsphingomyelin in bilayer membranes as revealed by 2H-NMR spectroscopy

Selectively deuterated N-palmitoyl sphingomyelins were studied by deuterium nuclear magnetic resonance spectroscopy ((2)H-NMR) to elucidate the backbone conformation as well as the interaction of the sphingolipids with glycerophospholipids. Macroscopic alignment of the lipid bilayers provided good spectral resolution and permitted the convenient control of bilayer hydration. Selective deuteration at the acyl chain carbons C(2) and C(3) revealed that the N-acyl chain performs a bend, similar to the sn-2 chain of the phosphatidylcholines. Profiles of C-D bond order parameters were derived from the segmental quadrupolar splittings for sphingomyelin alone and for sphingomyelin-phosphatidycholine mixtures. In the liquid-crystalline state, the N-acyl chain of sphingomyelin alone revealed significantly more configurational order than the chains of homologous disaturated or monounsaturated phosphatidylcholines. The average chain order parameters and the relative width of the order parameter distribution were correlated over a range of bilayer compositions. The temperature dependence of the (2)H-NMR spectra revealed phase separation in bilayers composed of sphingomyelin and monounsaturated phosphatidylcholine, in broad agreement with existing phase diagrams.     

Orientational order of cholesteryl oleate in low-density lipoprotein observed by 2H-NMR

Cholesteryl oleate, selectively deuterated at various positions along the acyl chain, has been incorporated into fresh human serum low-density lipoprotein (LDL2). Temperature-dependent 2H-NMR spectra were recorded between 15 and 45 degrees C. For deuterons at C-2' and C-5' of the acyl chain, two 2H-NMR spectral components, a broad and a narrow signal, are observed. This is interpreted as reflecting the coexistence of two cholesteryl ester regions in the LDL2 core which possess different degrees of order. The C-2H bond order parameters, SCD, are approx. 0.12-0.20 for the more ordered region and approx. 0.04-0.06 for the less ordered region. Longitudinal relaxation times, T1, of deuterated cholesteryl oleate are found to increase between C-8' and the terminal -C2H3 group, which is consistent with an increased rate of chain motion toward the free ends of the ester acyl chains.     

Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels.

Dioctadecyldimethylammonium bromide (DODAB) dispersions obtained by simply mixing the amphiphile in water, and by bath-sonication, were investigated by electron spin resonance (ESR) of stearic acids and their methyl ester derivatives, labeled at the 5th and 16th carbons of the acyl chain. The ESR spectra indicate that the non-sonicated dispersions are formed mainly by one population of DODAB vesicles, either in the gel (TT(m)) state. Around T(m) there is a co-existence of the two phases, with a thermal hysteresis of about 3.2 degrees C. In sonicated DODAB dispersions, spin labels indicate two different environments even for temperatures far below T(m): one similar to that obtained with non-sonicated samples, a gel phase, and another one in the liquid-crystalline state. The fluid phase domain present below T(m) could correspond to either the periphery of bilayer fragments, reported to be present in sonicated DODAB dispersions, or to high curvature vesicles.     

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.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.