Short-range interactions between lipid bilayers measured by X-ray diffraction

Interactions between lipid bilayers are critical in many biological processes in which membrane surfaces come close together. Recent X-ray diffraction analyses of bilayers subjected to known osmotic pressures have provided critical information on the magnitude of both the repulsive and the attractive forces that exist between phospholipid and glycolipid membranes.     

Dynorphin induced magnetic ordering in lipid bilayers as studied by P NMR spectroscopy

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T_m=24°C), as examined by ³¹P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T_m but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T_m. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T_m in parallel with the bilayer surface, because a single ³¹P NMR signal appeared at the perpendicular position of the ³¹P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T_m, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated ³¹P NMR lineshape.     

Electron diffraction studies of molecular ordering and orientation in phospholipid monolayer domains.

The molecular order and orientation of phase separated domains in monolayers of DP(Me)PE and DP(Me)2PE were determined by electron diffraction. Dark and bright fluorescent domains at the air-water interface were observed by fluorescence microscopy. The monolayers were transferred to Formvar coated electron microscope grids for electron diffraction studies. The positions of domains on the marker grids were recorded in fluorescence micrographs, which were used as guide maps to locate these domains in the electron microscope. Selected area electron diffraction patterns were obtained from predetermined areas within and outside the dark domains. Sharp hexagonal diffraction patterns were recorded from dark domains, and diffuse diffraction rings from bright areas in between dark domains. The diffraction results indicated that the dark domains and bright areas were comprised of lipid molecules in solid and fluid states, respectively. The orientation of diffraction patterns from adjacent locations within a dark domains changed gradually, indicating a continuous bending of the molecular packing lattice vector within these domains. Orientation directors in U-shaped DP(Me)2PE domains followed the turn of the arm; no vortex nor branching was indicated by electron diffraction. Directors branching from the "stem" of highly invaginated DP(Me)PE domains usually occurred at twinning angles of n pi/3 from the stem director, which would minimize packing defects in the development of thinner branches. Electron diffraction from local areas of individual domains proved that dark fluorescent domains were solid ones, and that pseudo-long range order existed in these solid domains.     

Secondary structure and lipid contact of a peptide antibiotic in phospholipid bilayers by REDOR

The chemical shifts of specific (13)C and (15)N labels distributed throughout KIAGKIA-KIAGKIA-KIAGKIA (K3), an amphiphilic 21-residue antimicrobial peptide, prove that the peptide is in an all alpha-helical conformation in the bilayers of multilamellar vesicles (MLVs) containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1). Rotational-echo double-resonance (REDOR) (13)C[(31)P] and (15)N[(31)P] experiments on the same labeled MLVs show that on partitioning into the bilayer, the peptide chains remain in contact with lipid headgroups. The amphipathic lysine side chains of K3 in particular appear to play a key role in the electrostatic interactions with the acidic lipid headgroups. In addition to the extensive peptide-headgroup contact, (13)C[(19)F] REDOR experiments on MLVs containing specifically (19)F-labeled lipid tails suggest that a portion of the peptide is surrounded by a large number of lipid acyl chains. Complementary (31)P[(19)F] REDOR experiments on these MLVs show an enhanced headgroup-lipid tail contact resulting from the presence of K3. Despite these distortions, static (31)P NMR lineshapes indicate that the lamellar structure of the membrane is preserved.     

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.     

Ca2+ interaction with phospholipid bilayers studied by multifrequency phase fluorometry

Calcium interaction with phospholipid membranes containing phosphatidic acid is studied by multifrequency phase fluorometry, using DPH as fluorescent molecule. DPH decay is analysed by a continuous distribution of lifetimes. The results suggest an increase of membrane heterogeneity at low calcium concentrations, without changes in the polarity of the environment surrounding the probe.     

Short range order of hydrocarbon chains in fluid phospholipid bilayers studied by x-ray diffraction from highly oriented membranes

We present a study of the short range ordering of hydrocarbon chains in phospholipid bilayers. The x-ray peak associated with the hydrocarbon chains has been probed by means of reciprocal space mappings. Using 20 keV undulator radiation and samples of negligible mosaicity (orientational disorder), the intensity distribution is probed as a function of two coordinates, the momentum transfer parallel and perpendicular to the bilayer, over a wide range and at high resolution. Structural results are obtained concerning the distribution of tilted segments, the correlation length and the radial distribution function of the quasi two-dimensional liquid structure. A comparison is made with published molecular dynamics data (H. Heller, M. Schaefer, and K. Schulten. 1993. J. Phys. Chem. 97:8343-8360) by direct Fourier transformation of the atomic coordinates. The exact prefactor in the relationship between interchain distance and peak position is derived.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Effect of ion-binding and chemical phospholipid structure on the nanomechanics of lipid bilayers studied by force spectroscopy

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.     

Effects of the peptide Magainin H2 on Supported Lipid Bilayers studied by different biophysical techniques

Given the increasing trend in bacterial antibiotic resistance, research on antimicrobial peptides and their mechanisms of action has become of huge relevance in the last years. Several studies have investigated the effects of a large variety of antimicrobial peptides directly on bacteria or on model lipid bilayers. In the case of model lipid bilayers, different systems are typically exploited; however, different results could be obtained due to the specific properties of the used system. Supported Lipid Bilayers and Giant Unilamellar Vesicles are among the most popular model systems. Here we used Atomic Force Microscopy and fluorescence microscopy to study the interaction of the antimicrobial peptide Magainin H2, an analog of Magainin 2 with increased hydrophobicity, on Supported Lipid Bilayers. We found that, for this kind of model bilayer, due to its strong interaction with the support, the lateral expansion of the membrane induced by the interaction with the peptides is initially inhibited and subsequently proceeds creating new bilayer regions with many defects. This scenario gives rise in Supported Lipid Bilayers to effects like initial increase of lateral pressure, formation of lipid tubes to release this increase, or development of bilayer regions with lower lipid density. Our results highlight that care should be given to the selected model system when studying and comparing the interaction of peptides with other lipid bilayer model systems.     

Grazing incidence X-ray diffraction of highly aligned phospholipid membranes containing the antimicrobial peptide magainin 2

We present the first study of grazing incidence X-ray diffraction on a model system of phospholipid membranes and antimicrobial peptides. For this purpose, highly oriented multilamellar samples have been prepared on solid substrates. By this technique, the short-range order of the lipid chains in the fluid L_α phase can be investigated quantitatively, including not only the mean distance between acyl chains, but also the associated correlation length. The short-range order in lecithin is found to be severely affected by the amphiphilic peptide magainin 2. Received: 7 June 1999 / Revised version: 6 September 1999 / Accepted: 17 September 1999     

Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t ₁-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid–water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid–water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid–lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.     

Hydrocarbon chain dynamics in lipid bilayers

A model is proposed for hydrocarbon chain dynamics in lipid bilayers. In the upper and middle parts of the chain all motion occurs by concerted rotations around at least two carbon carbon bonds at a time, preserving a structural with kinks (that is gauche^±trans gauche^? conformations) as the only deviations from the all-trans chain. At the end, independent rotations around carboncarbon bonds play a larger and larger part. This gives a reasonable interpretation of deuterium NMR data.     

Interaction of statins with phospholipid bilayers studied by solid-state NMR spectroscopy

Statins are drugs that specifically inhibit the enzyme HMG-CoA reductase and thereby reduce the concentration of low-density lipoprotein cholesterol, which represents a well-established risk factor for the development of atherosclerosis. The results of several clinical trials have shown that there are important intermolecular differences responsible for the broader pharmacologic actions of statins, even beyond HMG-CoA reductase inhibition. According to one hypothesis, the biological effects exerted by these compounds depend on their localization in the cellular membrane. The aim of the current work was to study the interactions of different statins with phospholipid membranes and to investigate their influence on the membrane structure and dynamics using various solid-state NMR techniques. Using ¹H NOESY MAS NMR, it was shown that atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and some percentage of pravastatin intercalate the lipid-water interface of POPC membranes to different degrees. Based on cross-relaxation rates, the different average distribution of the individual statins in the bilayer was determined quantitatively. Investigation of the influence of the investigated statins on membrane structure revealed that lovastatin had the least effect on lipid packing and chain order, pravastatin significantly lowered lipid chain order, while the other statins slightly decreased lipid chain order parameters mostly in the middle segments of the phospholipid chains.     

X-ray diffraction study of feline leukemia virus fusion peptide and lipid polymorphism

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using a temperature ramp with sequential X-ray diffraction. This peptide, the hydrophobic amino-terminus of p15E, has been proven to be fusogenic and to promote the formation of highly curved, intermediate structures on the lamellar liquid-crystal to inverse hexagonal phase transition pathway. The FeLV peptide produces marked effects on the thermotropic mesomorphic behaviour of MeDOPE, a phospholipid with an intermediate spontaneous radius of curvature. The peptide is shown to reduce the lamellar repeat distance of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism.     

The structure of lipid bilayers and the effects of general anaesthetics: An X-ray and neutron diffraction study

We have looked for the effects of three clinically used inhalational anaesthetics (nitrous oxide, halothane and cyclopropane) on the structure of lecithin/ cholesterol bilayers. The anaesthetics were delivered to the membranes in the gaseous phase, so that effects at clinical concentrations could be determined.High-resolution X-ray diffraction patterns were recorded out to 4 Å and analyzed using swelling experiments. Parallel neutron diffraction experiments were performed and analyzed using H₂O-²H₂O exchange. Methods were developed which enabled us to obtain confidence limits for the X-ray and neutron structure factors.The resultant X-ray and neutron scattering density profiles clearly define the positions of the principal molecular groups in the unperturbed bilayer. In particular, the high-resolution electron density profiles reveal features directly attributable to the cholesterol molecule. A comparison with the neutron scattering density profiles shows that cholesterol is anchored with its hydroxyl group at the water/hydrocarbon interface, aligned with the fatty acid ester groups of the lecithin molecule. We suggest that this positioning of the cholesterol molecule allows it to act as a thickness buffer for plasma membranes.In the presence of very high concentrations of general anaesthetics, the bilayers show increased disorder while maintaining constant membrane thickness. At surgical concentrations, however, there are no significant changes in bilayer structure at 95% confidence levels. We briefly review the literature previously used to support lipid bilayer hypotheses of general anaesthesia. We conclude that the lipid bilayer per se is not the primary site of action of general anaesthetics.     

Water orientation and motion in phospholipid bilayers: a comparison between 17O- and 2H-NMR.

17O- and 2H-NMR spectra were obtained of a lamellar phase of dipalmitoyl-3sn-phosphatidylcholine (DPL) and D2 17O with water content of 3--15 moles water/mole DPL, in the temperature range 20 to 80 degrees C. In every case, the quadrupole splittings observed for 17O were 6.6 times larger than those for 2H. Therefore the two methods contain in principle the same information, but with less details from 17O. On the other hand, dynamic information is easily obtained from 17O linewidth data and complements the deuterium results.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.