Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers.
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The water activity in dimyristoylphosphatidylcholine (DMPC) decreases by 60% when the lipid is dehydrated in the presence of trehalose concentrations higher than 0.02 M. In contrast, sucrose in concentrations 10 times higher produced only a 20% decrease in the water activity in the sample. Titrations of a DMPC solution in chloroform yielded 14 water molecules per lipid when pure water was added and seven water molecules per lipid when the titration was done with 0.025 M trehalose. The same concentrations of sucrose produced a turbid solution, which made it impossible to quantify the number of water molecules per lipid. Lipid monolayers spread on an air/water interface showed a decrease from 480 mV in pure water to 425 mV in 0.1 M trehalose. However, the same concentrations of sucrose produced an increase of less than 100 mV. Results obtained with Fourier transform infrared spectroscopy (FTIR) under the same conditions denoted that trehalose binds to the carbonyl groups, while sucrose showed no specific binding. It is concluded that per lipid molecule, 11 of 14 water molecules can be replaced by three trehalose molecules. About four are displaced by changes in the water activity of the bulk solution, and seven by specific interactions with the phospholipids. In this last case, at least two of them are linked to the carbonyls, and this appears to be the cause of the decrease in the dipole potential of the membrane. In contrast, four sucrose molecules displace only three water molecules per lipid, with no effect on the dipole potential or the carbonyl groups. (+info)
Dimyristoylphosphatidylcholine/C16:0-ceramide binary liposomes studied by differential scanning calorimetry and wide- and small-angle x-ray scattering.
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Ceramide has recently been established as a central messenger in the signaling cascades controlling cell behavior. Physicochemical studies have revealed a strong tendency of this lipid toward phase separation in mixtures with phosphatidylcholines. The thermal phase behavior and structure of fully hydrated binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and N-palmitoyl-ceramide (C16:0-ceramide, up to a mole fraction X(cer) = 0.35) were resolved in further detail by high-sensitivity differential scanning calorimetry (DSC) and x-ray diffraction. Both methods reveal very strong hysteresis in the thermal phase behavior of ceramide-containing membranes. A partial phase diagram was constructed based on results from a combination of these two methods. DSC heating scans show that with increased X(cer) the pretransition temperature T(p) first increases, whereafter at X(cer) > 0.06 it can no longer be resolved. The main transition enthalpy DeltaH remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to approximately 63 degrees C at X(cer) = 0.30. Upon cooling, profound phase separation is evident, and for all of the studied compositions there is an endotherm in the region close to the T(m) for DMPC. At X(cer) >/= 0.03 a second endotherm is evident at higher temperatures, starting at 32.1 degrees C and reaching 54.6 degrees C at X(cer) = 0.30. X-ray small-angle reflection heating scans reveal a lamellar phase within the temperature range of 15-60 degrees C, regardless of composition. The pretransition is observed up to X(cer) < 0.18, together with an increase in T(p). In the gel phase the lamellar repeat distance d increases from approximately 61 A at X(cer) = 0. 03, to 67 A at X(cer) = 0.35. In the fluid phase increasing X(cer) from 0.06 to 0.35 augments d from 61 A to 64 A. An L(beta')/L(alpha) (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5 degrees C) when X(cer) > 0.03. With cooling from temperatures above 50 degrees C we observe a slow increase in d as the coexistence region is entered. A sudden solidification into a metastable, modulated gel phase with high d values is observed for all compositions at approximately 24 degrees C. The anomalous swelling for up to X(cer) = 0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. (+info)
A thermodynamic study of the effects of cholesterol on the interaction between liposomes and ethanol.
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The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication. (+info)
Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid.
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Conformational reorganization of the amino-terminal four-helix bundle (22-kDa fragment) of apolipoprotein E (apoE) in binding to the phospholipid dimyristoylphosphatidylcholine (DMPC) to form discoidal particles was investigated by introducing single, double, and triple interhelical disulfide bonds to restrict the opening of the bundle. Interaction of apoE with DMPC was assessed by vesicle disruption, turbidimetric clearing, and gel filtration assays. The results indicate that the formation of apoE.DMPC discoidal particles occurs in a series of steps. A triple disulfide mutant, in which all four helices were tethered, did not form complexes but could release encapsulated 5-(6)-carboxylfluorescein from DMPC vesicles, indicating that the initial interaction does not involve major reorganization of the helical bundle. Initial interaction is followed by the opening of the four-helix bundle to expose the hydrophobic faces of the amphipathic helices. In this step, helices 1 and 2 and helices 3 and 4 preferentially remain paired, since these disulfide-linked mutants bound to DMPC in a manner similar to that of the 22-kDa fragment of apoE4. In contrast, mutants in which helices 2 and 3 and/or helices 1 and 4 paired bound poorly to DMPC. However, all single and double helical pairings resulted in the formation of larger discs than were formed by the 22-kDa fragment, indicating that further reorganization of the helices occurs following the initial opening of the four-helix bundle in which the protein assumes its final lipid-bound conformation. In support of this rearrangement, reducing the disulfide bonds converted the large disulfide mutant discs to normal size. (+info)
Pretransitional effects in dimyristoylphosphatidylcholine vesicle membranes: optical dynamometry study.
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We used micron-sized latex spheres to probe the phase state and the viscoelastic properties of dimyristoylphosphatidylcholine (DMPC) bilayers as a function of temperature. One or two particles were manipulated and stuck to a DMPC giant vesicle by means of an optical trap. Above the fluid-gel main transition temperature, T(m) congruent with 23.4 degrees C, the particles could move on the surface of the vesicle, spontaneously (Brownian motion) or driven by an external force, either gravity or the laser beam's radiation pressure. From the analysis of the particle motions, we deduced the values of the membrane hydrodynamic shear viscosity, eta(s), and found that it would increase considerably near T(m). Below T(m), the long-distance motion of the particles was blocked. We performed experiments with two particles stuck on the membrane. By optical dynamometry, we measured the elastic resistance of the membrane to a variation in the interparticle distance and found that it would decrease considerably (down to zero) when the temperature was increased to T(m). We propose an interpretation relating the elastic response to the membrane curvature modulus, k(C). In this scheme, the two-bead dynamometry experiments provide a direct measurement of k(C) in the P'(beta) phase of lipid bilayers. (+info)
A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study.
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Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305). (+info)
Rotational mobility and orientational stability of a transport protein in lipid membranes.
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A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid. (+info)
Oriented circular dichroism of a class A amphipathic helix in aligned phospholipid multilayers.
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The effect of lipid phase state on the orientation and conformation of a class A alpha-helical peptide on aligned lipid multilayers was examined using oriented circular dichroism spectroscopy. A comparison of oriented spectra in aligned peptide-lipid multilayers with CD spectra of unaligned peptide lipid vesicle complexes is consistent with a preferential alignment of helices parallel to the membrane surface at temperatures above and below the main acyl-chain melting transition temperature of the phospholipid. Changes are observed in the oriented CD spectra with lipid phase state which are attributed to a subtle conformational change of the peptide on the lipid surface. The results are compared with available experimental data on membrane-active lytic and antimicrobial helical peptides. (+info)