Lipid Chain-melting Transition Research Articles

Membrane Thinning Is Not A Unique Signal Of Pore Formation By Antimicrobial Peptides

We have observed a hydrocarbon chain length dependent perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) using X-ray diffraction, solid-state 2H-NMR, differential scanning calorimetry and dilatometry. In the gel phase, PGLa assumes a surface alignment and induces a quasi-interdigitated phase, previously reported also for other peptides. This effect is most pronounced for C18 phosphatidylglycerol. Above the lipid chain melting transition, in the fluid phase, the PGLa helix inserts into the membrane above a certain threshold concentration. In this case 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 in order 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.

Temperature-Dependent Transmembrane Insertion of the Amphiphilic Peptide PGLa in Lipid Bilayers Observed by Solid State 19F NMR Spectroscopy

The alignment of the antimicrobial peptide PGLa in a lipid bilayer was characterized by solid state 19F NMR on selectively CF3-labeled peptides in oriented samples. Previous studies in liquid crystalline model membranes had shown that the amphiphilic α-helix of PGLa adopts a surface-aligned S-state or a tilted T-state, depending on the peptide/lipid ratio. Only in the presence of the synergistic partner peptide magainin 2 had PGLa been found insert into the bilayer in a transmembrane I-state orientation. Here, we have characterized the peptide alignment but as a function of temperature and lipid phase state. At temperatures below the lipid chain melting transition, PGLa is now seen to be able to insert on its own, with its helical axis nearly parallel to the bilayer normal (tilt angle of ∼170°), forming the I-state. Above the lipid phase transition, PGLa is aligned in the known T-state (tilt angle of ∼130°), but it is found flip into the S-state at more elevated temperatures (tilt angle of ∼96°). This way...

Thermal assembly of a biomimetic mineral/collagen composite

A strategy is described for exploiting temperature driven self-assembly of collagen and thermally triggered liposome mineralization to form a mineralized collagen composite from an injectable precursor fluid. Optical density and rheological experiments demonstrated the formation of a collagen gel when acid-soluble type I collagen solutions (1–7 mg/ml) were heated to 24–30°C. Scanning calorimetry experiments demonstrated that mixtures of calcium- and phosphate-loaded liposomes composed of dipalmitoylphosphatidylcholine (90 mol%) and dimyristoylphosphatidylcholine (10 mol%) were stable at room temperature but formed calcium phosphate mineral when heated above 35°C, a consequence of the release of entrapped salts at the lipid chain melting transition. The formation of calcium phosphate mineral induced by triggered release of calcium and phosphate was detected as an endothermic transition (Δ H=6.2±1.1 kcal/mol lipid) near the lipid chain melting transition ( T m=37°C). Combining an acid-soluble collagen solution with calcium- and phosphate-loaded liposomes resulted in a liposome/collagen precursor fluid, which when heated from room temperature to 37°C formed a mineralized collagen gel. The dynamic storage modulus of the collagen scaffold increased upon mineralization, and direct nucleation of mineral from the collagen scaffold was detected by electron microscopy.

Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.

Thermally Triggered Calcium Phosphate Formation from Calcium-Loaded Liposomes

A thermally triggered liposome-based mineralization system is described that is metastable at ambient temperature but rapidly forms calcium phosphate mineral upon warming to physiologic temperature. Mixing of a calcium-loaded lipid vesicle suspension with aqueous inorganic phosphate resulted in a stable liposome suspension whose bulk ionic concentration was highly supersaturated with respect to hydroxyapatite and other calcium phosphate minerals. The mineralization activity of metastable liposome suspensions was found to be strongly dependent both on temperature and on the composition of the phospholipids that comprise the vesicle membrane. No detectable mineral formation occurred in the metastable liposome suspensions during storage for several weeks at room temperature. However, when the liposome suspensions were heated to near the lipid chain melting transition (Tm), Ca 2+ was released from the lipid vesicles and reacted with extravesicular phosphate to form calcium phosphate mineral. Infrared spectroscopy and powder X-ray diffraction analysis of the reaction products indicated the formation of both apatite and brushite minerals, a finding that is consistent with the changing pH conditions of thermally triggered mineralization. Mixtures of miscible phospholipids (dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine) were used to tailor Tm to physiologic temperature. This strategy was employed for the preparation of metastable liposome suspensions that were stable for long periods of time at room temperature but that mineralized rapidly when heated to 37 °C. The potential medical and dental significance of thermally triggered liposomal mineralization is discussed.

Effect of organotin compounds on membrane lipids: Fluorescence spectroscopy studies

The temperature dependence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) fluorescence anisotropy has been studied to investigate the influence of mono-n-butyltin chloride (MBTC), di-n-butyltin chloride (DBTC) and tri-n-phenyl chloride (TPTC) on the physicochemical state of dipalmitoyl phosphatidylcholine. Below the lipid chain melting transition (Tm), fluorescence anisotropy values of DPH and TMA-DPH are increased by the presence of the organotins, without important modifications of the phase transition temperature. A possible difference in localization of the organotin compounds is suggested by the differential effect of the probes. It is suggested that there is localization in the hydrophobic core of the bilayer for TPTC, and at the head-group level for DBTC, and a homogeneous distribution in the bilayer for MBTC. Similar studies have been performed in liposomal suspensions of cardiolipin, phosphatidylserine and egg phosphatidylcholine.

Peptides modeled on the transmembrane region of the slow voltage-gated IsK potassium channel: structural characterization of peptide assemblies in the beta-strand conformation.

A 27-residue peptide, having a sequence corresponding to the transmembrane domain of the IsK protein with slow voltage-gated potassium channel activity, has been incorporated into synthetic saturated-chain phospholipid membranes. The peptide-lipid complexes have been characterized by attenuated-total-reflection Fourier-transform-infrared spectroscopy (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, 31P and 2H nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and low-angle X-ray diffraction. From FTIR spectroscopy, it is found that, when reconstituted into membranes by dialysis from 2-chloroethanol, the peptide has a predominantly beta-strand secondary structure in which the peptide backbone is oriented at an angle of approximately 56 degrees relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes the transition to the fluid phase. The ESR and NMR spectra from spin-labeled and 2H-labeled phospholipids, respectively, indicate that the incorporated peptide restricts the rotational motion of the lipids, without appreciably affecting the chain order, in a way similar to that found for integral membrane proteins. The characteristic two-component ESR spectra from spin-labeled lipids further indicate a selectivity in the interaction of anionic phospholipids with the peptide. The motional restriction of the chains of the spin-labeled phosphatidylcholine and the reduction in the enthalpy of the lipid chain-melting transition indicate that, on average, approximately two to three phospholipid molecules interact directly with each peptide monomer, which is consistent with a limited degree of aggregation of the beta-sheet structures. Both 31P NMR spectroscopy and X-ray diffraction indicate that the lipid-peptide complexes have a lamellar structure up to the highest peptide concentration studied (Rp = 0.2). The surface area occupied by lipid molecules (ca. 30 A2 per chain) in the peptide complexes, deduced from the lamellar repeat spacings at defined water content, is very similar to that in pure fluid lipid bilayers, consistent with the 2H NMR results. The additional membrane surface area contributed by the peptide is approximately 112 A2 per monomer. This large value for the peptide area in the fluid bilayer is consistent with the ATR studies of dry peptide/lipid films which suggest that the long axis of the beta-strand is strongly tilted with respect to the bilayer normal (56 degrees in the dry film).

Interaction of alpha-lactalbumin with phosphatidylglycerol. Influence of protein binding on the lipid phase transition and lipid acyl chain mobility.

The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes.

2H-NMR investigation of DMPC/glycophorin bilayers

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the phase equilibria, and the temperature and concentration dependences of the phospholipid hydrocarbon chain order, of mixtures of glycophorin in dimyristoylphosphatidylcholine. In the fluid phase it is found that the protein has only a slight effect on the first moment of the 2H spectrum, which for perdeuterated chains is a direct measure of the average chain orientational order. However, analysis of the rate of change of the first moment with respect to protein concentration, at different temperatures within the fluid phase, shows that at a molar protein concentration of about 0.0295 ± 0.01, the lipid chain order (or M 1) is essentially independent of temperature. At this concentration the chain order is determined by the lipid's interaction with the protein and one can conclude that about 34 (±12) lipids are required to solvate the protein. At higher lipid concentrations these lipids are freely exchanging, on the NMR time scale, with the other lipids in the bilayer. At glycophorin concentrations below about 1 mol% there is a two-phase coexistence region at temperatures below the pure lipid's chain melting transition. The boundary between the fluid phase and this two-phase region curves downwards (is concave downwards), whereas the boundary between the two-phase region and the gel phase, while naturally occurring at lower temperatures than the upper boundary, is concave upwards. As a consequence the protein partitions preferentially into the fluid phase. This behaviour is similar to that observed in a number of other protein/lipid and peptide/lipid mixtures where it was suggested that those systems may have been close to a critical mixing point and some characteristics of a continuous phase change were noted. Indeed, at glycophorin concentrations near and above 1 mol% there are indications that the phase behaviour becomes more complex, suggesting the presence of significant protein/protein interactions and that this system may be close to a critical point.

Hydration of DOPC bilayers by differential scanning calorimetry

The phase diagram of the unsaturated lipid dioleoylphosphatidylcholine (DOPC) in aqueous multibilayer dispersions has been constructed from a series of differential scanning calorimetry (DSC) thermograms over the temperature range from −40 to +10° C , covering a range of hydration levels from the monohydrate to excess free water. Both the lipid chain melting transition and the ice melting point are found to be hydration dependent. From their respective variations it is found that the bilayer in the gel phase binds approximately 9 H 2O per lipid, while the liquid-crystalline state has a saturation limit near 20 H 2O. The water transition exhibits a hydration-dependent melting point depression, which can be explained in terms of newly incorporated water between the bilayer surfaces upon melting of the acyl chains, and which is reminiscent of the events that occur at the pre-transition for saturated lipids. From the melting point depression, the thermodynamic activity of the interbilayer water can be calculated and thus the repulsive hydration force characterized quantitatively. We evaluate a (non-isothermal) hydration force decay constant around 2.8 H 2O, which demonstrates that this DSC approach is well-suited for quantitatively characterizing the hydration properties of unsaturated lipid dispersions at low temperature.

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.

Dynamics of phospholipid tubules in a concentrated solution: Results from high-field magnetic birefringence and quasielastic light scattering.

The dynamical diffusion of phospholipid tubules in highly concentrated solution has been studied as a function of temperature by high-field magnetic birefringence and by dynamic light scattering in zero field. Data at a temperature well below the lipid chain-melting transition T=${\mathit{T}}_{\mathit{m}}$ are analyzed using theoretical predictions for rigid-rod dynamics. While qualitatively successful, these lead to an estimate of the rotational diffusion constant which is larger than that anticipated when dynamical rod entanglement in a homogeneous solution is the only effect considered at high concentration. For T\ensuremath{\rightarrow}${\mathit{T}}_{\mathit{m}}$, the results from both experimental techniques exhibit a marked temperature dependence, revealing dynamical studies to be a sensitive and potentially interesting probe of the phase transformation at ${\mathit{T}}_{\mathit{m}}$.

Polymorphism of the bilayer membranes in the ordered phase and the molecular origin of the lipid pretransition and rippled lamellae

The lamellar-to-undulated-lamellar phase transition (L β′ →P β′, pretransition) in lipid bilayers is shown to be a phenomenon which exists only if lipid polar headgroups are sufficiently hydrated and if the interchain packing is sufficiently weak. The minimal lipid hydrophilicity and the critical amount of the lipid-bound water can be related to the lipid chain-melting transition temperature; the latter must not exceed some maximal, chainlength-dependent value if the pretransition is to exist. The minimally required amount of the lipid-bound water itself is essentially chainlength independent, however, and unaffected by the method of hydration variation: physical dehydration, hydrational competition between the lipid molecules and the substances dissolved in the aqueous subphase, or decreasing lipid headgroup polarity all affect the pretransition temperature similarly on the appropriate scale. Simple, phenomenological expressions for the evaluation of the bilayer subtransition, pretransition and chain-melting phase transition temperature as a function of the lipid chainlength are presented. They show that, even in excess water, bilayers will tend to undulate only as long as each of the two identical lipid chains will contain between 12 ± 1and22 ± 1 carbon atoms the P β-phase region for the less polar lipids being as a rule narrower. To get a theoretical means for quantitatively studying the effects of the lipid hydration on the bilayer pretransition, an interaction-balance method is proposed for describing undulated membranes at the molecular level. This is based on comparing the free-energy gain from the increased headgroup hydration with the free-energy loss caused by the reduced chain-chain attraction upon ripple formation. A rationale is thus found for scaling the pretransition temperature in terms of the hydration-induced chain-melting phase transition shift or of the lipid surface hydrophilicity (see Fig. 8) Within the framework of such a model the recently reported (de)hydration dependence of the bilayer-undulation period is reproduced with reasonable accuracy. Furthermore, it is estimated that at least 12 ± 2 water molecules must be associated with each lipid head for the bilayer undulation to be feasible. The closer the system is to this boundary condition the longer is the repeat-distance for the surface undulations and the less stable is the undulated bilayer phase.

Interaction of insulin receptors with lipid bilayers and specific and nonspecific binding of insulin to supported membranes

In the first part, we study the interaction of the insulin receptor with model membranes of dimyristoylphosphatidylcholine (DMPC) by various techniques, including calorimetry, densitometry, static light scattering, and electron microscopy. By analyzing the pronounced depression of the lipid chain melting transition in terms of the Van Laar-Hildebrand theory of regular dilute solutions, an (exothermic) interaction energy of Wp = 2000 kJ.mol-1 is found for the receptor and of WL = 0.6 kJ.mol-1 for the lipid. This is interpreted in terms of an adsorption of the 2 hydrophilic head groups of the receptor to the membrane surface so that 1 protein interacts with about 2000 lipids. This number is verified by freeze-fracture electron microscopy. Binding of insulin induces a remarkable decoupling of the receptor head group from the membrane, pointing to a pronounced conformational change. In the second part, we introduce a simple fluorescence technique by which adsorption isotherms of water-soluble and fluorescent-labeled substrates, such as insulin, to membranes may be determined. It is based on the selective evanescent field excitation of ligands adsorbed to supported planar bilayers on argon-sputtered glass plates. These are deposited by the monolayer transfer technique or by vesicle condensation. The reconstituted receptor exhibits a weak (binding constant Kw = 3 X 10(9) L.M-1) and a strong (binding constant Ks greater than 10(10) L.M-1) binding site. Insulin exhibits a weak but remarkable nonspecific binding to bilayers of pure DMPC and DMPC containing 20% positively charged lipid and a strong binding to DMPC containing negatively charged lipids such as phosphatidylserine or ganglioside (GT1b).(ABSTRACT TRUNCATED AT 250 WORDS)

Availability of dinitrophenylated lipid haptens for specific antibody binding depends on the physical properties of host bilayer membranes.

We have measured the binding of two radioiodinated monoclonal anti-dinitrophenyl antibodies (IgE and IgG2a) to two dinitrophenylated lipid haptens in lipid bilayer membranes having various compositions and physical properties. These antibodies bind strongly to the lipophilic dinitrophenyl group in some membranes. Dimyristoylphosphatidylcholine and dipentadecanoylphosphatidylcholine containing 2 mol % dinitrophenyl lipid hapten bind anti-dinitrophenyl antibodies below the chain-melting transition temperatures of these lipids (22 and 35 degrees C, respectively) but not above these temperatures. Evidently, the lipophilic dinitrophenyl group is partially or completely buried in the hydrophobic region of these bilayers at temperatures above the chain-melting transition temperatures. The inclusion of increasing concentrations of cholesterol in such membranes (e.g. in dimyristoylphosphatidylcholine at 37 degrees C) results in a marked enhancement of antibody binding. It was found that a third lipid hapten containing the dinitrophenyl group does not show this strong dependence of antibody binding on the physical state of the lipid membrane. The weak immunologic degranulation of rat basophil leukemia cells by dimyristoylphosphatidylcholine membrane targets at 37 degrees C can be attributed to a weak binding of anti-dinitrophenyl IgE to these membranes (Balakrishnan, K., Hsu, F. J., Cooper, A. D., and McConnell, H. M. (1982) J. Biol. Chem. 257, 6427-6433). However, if the antibody is first allowed to bind to this membrane below the lipid chain-melting transition temperature, these IgE-coated membrane targets are very effective in releasing serotonin from the rat basophil leukemia cells when the temperature is raised to 37 degrees C.

The interaction of apoprotein from porcine high-density lipoprotein with dimyristoyl phosphatidylcholine.

1 The interactions between the lipid and protein in complexes of dimyristoyl lecithin and porcine apoprotein were studied using electron spin resonance and fluorescent probes, dissolved in the lipid and covalently bonded to the protein. 2 The chain-melting transition of dimyristoyl lecithin is broadened on complexing with porcine apoprotein, indicating that the cooperative interaction between the lipid hydrocarbon chains is modified in the complex. 3 The broadening of the chain-melting transition is seen to be greatest by probes located at or near the glycerol region of the phospholipid. 4 Labels covalently bound to the protein experience only a small perturbation from the lipid chain-melting transition. 5 The results are interpreted as being consistent with a model for the complex, consisting of a lipid-bilayer, partially coated with protein and with some interdigitation of the protein.