We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly ...asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the
-chain penetrate deeper into the opposing leaflet by half a CH
group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.
The applicability of full‐q‐range models to fit low‐resolution X‐ray diffraction data from multibilayers exhibiting only weak quasi‐Bragg peak scattering has been analysed. The models consider ...different structure factors, accounting for different types of lattice disorder caused by stacking faults or bending fluctuations. Numerical tests of the models, considering instrumental influence of a line‐focus collimation system, demonstrated that Bragg peak line shapes given by different lattice disorders cannot be discerned. However, line‐shape parameters can be determined for a particular sample, if the type of disorder is known a priori. This has been verified by comparing the experimental results for the fluctuation parameter of palmitoyl‐oleoyl phosphatidylcholine (POPC) as a function of temperature with high‐resolution data on the same lipid. Tests further show that the calculation of structural parameters, such as the membrane thickness or the extent of the interbilayer water region, is not obscured by the smearing imposed by the instrument. The model was further applied successfully to experimental data of lipid mixtures composed of sphingomyelin (SM)/POPC/cholesterol and dipalmitoyl phosphatidylethanolamine (DPPE)/dipalmitoyl phosphatidylglycerol (DPPG). The structural parameters determined give valuable insight into the physical state of the membrane system, which is not accessible when quasi‐Bragg reflections only are considered.
We have studied the phase behavior of binary mixtures of long- and short-chain lipids, namely, dimyristoyl phosphatidylcholine (DMPC) and dihexanoyl phosphatidylcholine (DHPC), using optical ...microscopy and small-angle neutron scattering. Samples with a total lipid content of 25 wt %, corresponding to ratios Q (DMPC/DHPC) of 5, 3.2, and 2, are found to exhibit an isotropic (I) --> chiral nematic (N) --> lamellar phase sequence on increasing temperature. The I-N transition coincides with the chain melting transition of DMPC at Q = 5 and 3.2, but the N phase forms at a higher temperature for Q = 2. All three samples form multilamellar vesicles in the lamellar phase. Our results show that disklike "bicellar" aggregates occur only in the lower temperature isotropic phase and not in the higher temperature magnetically alignable N phase, where they were previously believed to exist. The N phase is found to consist of long, flexible wormlike micelles, their entanglement resulting in the very high viscosity of this phase.
The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was ...studied by differential scanning calorimetry, x-ray diffraction,
31P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (L
c′) to gel (L
β
) phase transition and the higher temperature endotherm to a L
β
to lamellar liquid-crystalline (L
α
) phase transition. At the L
c′/L
β
phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from ∼11
Å to ∼50
Å, and this value decreases slightly at the L
β
/L
α
phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8
Å to 43.5
Å at the L
c′/L
β
phase transition, consistent with the loss of the hydrocarbon chain tilt of ∼12°, and decreases to 37.8
Å at the L
β
/L
α
phase transition. The calculated cross-sectional areas of the TMCL molecules are ∼79
Å
2 and ∼83
Å
2 in the L
c′ and L
β
phases, respectively, and we estimate a value of ∼100
Å
2 in the L
α
phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L
c′ phase, TMCL molecules possess tilted all-
trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L
β
phase the untilted, all-
trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L
α
phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L
c′ to the L
β
and then to the L
α
phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our
31P-NMR results indicate that although the polar headgroup retains some mobility in the L
c′ phase, its motion is much more restricted in the L
β
and especially in the L
α
phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes.
Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this ...method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.
One persistent puzzle in the life sciences is the asymmetric lipid composition of the cellular plasma membrane: while the exoplasmic leaflet is enriched in lipids carrying predominantly saturated ...fatty acids, the cytoplasmic leaflet hosts preferentially lipids with (poly‐)unsaturated fatty acids. Given the high energy requirements necessary for cells to maintain this asymmetry, the question naturally arises regarding its inherent benefits. In this paper, we propose asymmetry to represent a potential solution for harmonizing two conflicting requirements for the plasma membrane: first, the need to build a barrier for the uncontrolled influx or efflux of substances; and second, the need to form a fluid and dynamic two‐dimensional substrate for signaling processes. We hence view here the plasma membrane as a composite material, where the exoplasmic leaflet is mainly responsible for the functional integrity of the barrier and the cytoplasmic leaflet for fluidity. We reinforce the validity of the proposed mechanism by presenting quantitative data from the literature, along with multiple examples that bolster our model.
The lipid composition of the two plasma membrane leaflets is highly asymmetric. Here we speculate whether lipid asymmetry reflects nature's need for harmonizing conflicting demands. We assign two plasma membrane functions to the two leaflets: the outer leaflet provides resistance to hydrophilic substances, and the inner leaflet fluidity to signaling molecules.
One persistent puzzle in the life sciences is the asymmetric lipid composition of the cellular plasma membrane: while the exoplasmic leaflet is enriched in lipids carrying predominantly saturated ...fatty acids, the cytoplasmic leaflet hosts preferentially lipids with (poly-)unsaturated fatty acids. Given the high energy requirements necessary for cells to maintain this asymmetry, the question naturally arises regarding its inherent benefits. In this paper, we propose asymmetry to represent a potential solution for harmonizing two conflicting requirements for the plasma membrane: first, the need to build a barrier for the uncontrolled influx or efflux of substances; and second, the need to form a fluid and dynamic two-dimensional substrate for signaling processes. We hence view here the plasma membrane as a composite material, where the exoplasmic leaflet is mainly responsible for the functional integrity of the barrier and the cytoplasmic leaflet for fluidity. We reinforce the validity of the proposed mechanism by presenting quantitative data from the literature, along with multiple examples that bolster our model.
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