Properties Of Bilayer Membranes Research Articles

Cooperative dynamics in a model DPPC membrane arise from membrane layer interactions

The dynamics of model membranes can be highly heterogeneous, especially in more ordered dense phases. To better understand the origins of this heterogeneity, as well as the degree to which monolayer systems mimic the dynamical properties of bilayer membranes, we use molecular simulations to contrast the dynamical behavior of a single-component dipalmitoylphosphatidylcholine (DPPC) lipid monolayer with that of a DPPC bilayer. DPPC is prevalent in both biological monolayers and bilayers, and we utilize the widely studied MARTINI model to describe the molecular interactions. As expected, our simulations confirm that the lateral structure of the monolayer and bilayer is nearly indistinguishable in both low- and high-density phases. Dynamically, the monolayer and bilayer both exhibit a drop in mobility for dense phases, but we find that there are substantial differences in the amplitude of these changes, as well as the nature of molecular displacements for these systems. Specifically, the monolayer exhibits no apparent cooperativity of the dynamics, while the bilayer shows substantial spatial and temporal heterogeneity in the dynamics. Consequently, the dynamical heterogeneity and cooperativity observed in the bilayer membrane case arises in part due to interlayer interactions. We indeed find a substantial interdigitation of the membrane leaflets which appears to impede molecular rearrangement. On the other hand, the monolayer, like the bilayer, does exhibit complex non-Brownian molecular displacements at intermediate time scales. For the monolayer system, the single particle motion can be well characterized by fractional Brownian motion, rather than being a consequence of strong correlations in the molecular motion previously observed in bilayer membranes. The significant differences in the dynamics of dense monolayers and bilayers suggest that care must be taken when making inferences about membrane dynamics on the basis of monolayer studies.

Colloidal Apatite Nanoparticles: Insights on their Interaction with Cells and Artificial Lipid Membranes

Biomimetic nanocrystalline apatites are analogous to bone mineral. They can be exploited not only for bone regeneration applications, but it is also possible to take advantage of their biomimetic features to explore novel domains of research such as in nanomedicine, if the nanoparticles are stabilized as a colloidal formulation. In this contribution, we concentrate on AEP/HMP-stabilized colloidal apatite nanoparticles (NPs) and on their interaction with different types of cells so as to get experimental evidence on their low cytotoxicity, non-proinflammatory potential, and good compatibility with Red Blood Cells. We then started to explore their interaction with an artificial free-standing phospholipid bilayer, as a simplified model for cell membranes: results indicate, for the first time, that these colloidal apatite NPs can modulate phospholipid bilayer membrane properties, and may even favor the permeation of small molecules (illustrated here with luminescent FITC), which could ultimately be exploited for nanomedicine applications in view of enhancing intracellular drug delivery.

Influence of Glutamic Acid Residues on the Properties of Model Membrane-Spanning Helices

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- amide) is a constructive model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J.Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged glutamic acid side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments showed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 9.0, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The E16-containing peptide seems to exhibit multi-state behavior at pH 10.9, in bilayers formed by ether-linked lipids, suggesting a pKa that is above pH 9 for the E16 side chain. The rather modest shift in the 2H quadrupolar splittings suggests that the orientation of the transmembrane peptide helix changes rather little at high pH. It is conceivable that the close proximity of E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the pKa of E16. The molecular cousin having E14 instead of E16 shows multi-state behavior from pH 4.0 to pH 10.9 rendering pKa determination enigmatic at this time. Some puzzles remain with respect to comparisons between E16 and E14. We are additionally investigating the peptide-lipid behavior when Glu is introduced in position 12, at the peptide center.

Influence of pH and Side-Chain Negative Charge on the Behavior of Designed Transmembrane Peptides in Lipid Bilayers

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a favorable model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments were used to examine the peptide orientation and dynamics as functions of the lipid bilayer thickness and pH in hydrated lipid bilayer membranes. We observed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 8.2, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The glutamic acid residue, though protonated, seemed to confer multi-state behavior at pH 2.5, and the resulting populations exhibited slow exchange on the NMR time scale. The deprotonation of E16 at pH 8.2 did not have any effect on the peptide orientation, perhaps suggesting that the close proximity of E16 to W19 (on the next helical turn) could provide stability to the peptide helix. We are also studying the peptide-lipid behavior when Glu is substituted in position 12 and/or 14, individually or together.

Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes.

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.

Effects of Sphingosine 2N- and 3O-Methylation on Palmitoyl Ceramide Properties in Bilayer Membranes

To study the role of the interfacial properties of ceramides in their interlipid interactions, we synthesized palmitoylceramide (PCer) analogs in which a methyl group was introduced to the amide-nitrogen or the C3-oxygen of the sphingosine backbone. A differential scanning calorimetry analysis of equimolar mixtures of palmitoylsphingomyelin (PSM) and PCer showed that these sphingolipids formed a complex gel phase that melted between 67°C and 74°C. The PCer analogs also formed gel phases with PSM, but they melted at lower temperatures compared with the system with PCer. In complex bilayers composed of an unsaturated glycerophospholipid, PSM, and cholesterol, the 3O-methylated ceramide formed a cholesterol-poor ordered phase with PSM. However, the 2N-methylated and doubly methylated (2N and 3O) PCer analogs failed to displace sterol from interactions with PSM. Like PCer, the analogs reduced sterol affinity for the complex bilayers, but this effect was most pronounced for the 3O-methylated ceramide. Taken together, our results show that 2N-methylation weakened the ceramide-PSM interactions, whereas the 3O-methylated ceramide behaved more like PCer in interactions with PSM. Our findings are compatible with the view that interlipid interactions between the amide-nitrogen and neighboring lipids are important for the cohesive properties of sphingolipids in membranes, and this also appears to be a valid model for ceramide.

Membrane properties of and cholesterol's interactions with a biologically relevant three-chain sphingomyelin: 3O-palmitoyl-N-palmitoyl-D-erythro-sphingomyelin

Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5°C (ΔH 8kcal/mol), which is about 15°C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.

The effects of N-acyl chain methylations on ceramide molecular properties in bilayer membranes

Long-chain saturated ceramides possess the ability to form gel domains in fluid bilayer membranes. Such domains may also contain sphingomyelin, but generally exclude cholesterol. We studied the effect of N-acyl chain methylations on the ability of ceramide to form ceramide- and sphingomyelin-containing gel domains that displace sterol. Fluorescence quenching of probes displaying different lateral partitioning in heterogeneous lipid bilayers showed that the methyl branches induced position-dependent changes in the lateral distribution of the ceramides. Distally monomethylated ceramides interacted with sphingomyelin and displaced sterol, whereas proximally monomethylated and polymethylated ceramides appeared to be located outside of sterol/sphingomyelin-enriched domains. The branched ceramides also markedly reduced the bilayer affinity for sterol as determined from the equilibrium partitioning of sterol between lipid vesicles and cyclodextrin. Altogether, alterations in intermolecular interactions induced by the methyl branches markedly affected the molecular properties of ceramide in artificial bilayers.

The Effects of Long-Chain Base Methylations on Ceramide Molecular Properties in Bilayer Membranes

Structural modifications have position-dependent effects on the molecular properties of ceramides in bilayer membranes. Ceramides with additional chemical groups, such as triple bonds or cyclic structures, in the long-chain base near the head group region induce similar thermal stabilization and displacement of sterol from sphingomyelin (SM)-enriched domains as unmodified ceramides1. However, the ability to thermally stabilize bilayers is significantly altered for ceramides with varying acyl chain length2. In addition, we have shown that methyl-branches in the amide-linked acyl chain markedly alter the lateral distribution of ceramides in bilayers, affecting the ability of the ceramides to interact with SM to form gel-phases that exclude sterol3. This study aims to determine the effects of yet another kind of structural modification on ceramide molecular properties, namely introduction of a methyl-group either to the amide-link or the hydroxyl-group of the sphingosine base, or both (yielding NMeCer, OMeCer, and NMeOMeCer, respectively). The analogs were prepared by enzymatic release of ceramides from corresponding N- or O-methylated SMs, and their membrane properties were studied with different fluorescence based methods. The ability of the analogs to interact with SM to form gel-phase domains, and the possible displacement of sterol from such domains was determined from the quenching susceptibility of trans-parinaric acid in gel phases, and cholestatrienol in sterol-enriched domains. In addition, the overall affinity of sterol for bilayers containing the ceramide analogs was determined from an equilibrium distribution of cholestatrienol between the bilayers and methyl-β-cyclodextrin. The results are discussed in relation to possible ceramide functions in cell membranes. 1Megha et al., 2007 Biochim Biophys Acta 1768: 2205-12. 2Nybond et al., 2005 Biochim Biophys Acta 1718: 61-6. 3Maula et al., Chem Phys Lipids 163S1: S2-3, 2010.

1A1436 圧力摂動熱量測定および密度測定によるエステルおよびエーテル結合型リン脂質二分子膜の体積挙動の解明(生体膜・人工膜1(構造・物性、ダイナミクス),第49回日本生物物理学会年会)

1A1436 圧力摂動熱量測定および密度測定によるエステルおよびエーテル結合型リン脂質二分子膜の体積挙動の解明(生体膜・人工膜1(構造・物性、ダイナミクス),第49回日本生物物理学会年会)

Analytical derivation of thermodynamic properties of bilayer membrane with interdigitation

We consider a model of bilayer lipid membrane with interdigitation, in which the lipid tails of the opposite monolayers interpenetrate. The interdigitation is modeled by linking tails of the hydrophobic chains in the opposite monolayers within bilayer as a first approximation. This model corresponds to the types of interdigitation that are not related with the areal “hydrophobic” dilation of the membrane. A number of essential thermodynamical characteristics are calculated analytically and compared with the ones of a regular bilayer membrane without interdigitation. Important difference between lateral pressure profiles at the layers interface for linked and regular bilayer models is found. In the linked case, the lateral pressure mid-plane peak disappears, while the entropy decreases and the free energy per chain increases. Within our model we found that in case of elongation of the chains inside a nucleus of, e.g., liquid-condensed phase, homogeneous interdigitation would be more costly for the membrane’s free energy than energy of the hydrophobic mismatch between the elongated chains and the liquid-expanded surrounding. Nonetheless, an inhomogeneous interdigitation along the nucleus boundary may occur inside a “belt” of a width that varies approximately with the hydrophobic mismatch amplitude.

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes ⩾ 4 · 10 −5 . A minimal concentration of about 6 · 10 11 dipicrylamine ions per cm 2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method N t opt is compared with the concentrations N t el obtained from simultaneous electrical relaxation measurements. N t opt and N t el agreed at low dipicrylamine concentrations (10 −8–10 −7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10 −6 M). In the saturation range N t opt was maximally four times higher than N t el . The significance of this difference is discussed together with general aspects of the saturation phenomenon.