Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

A theory of the electric field-induced phase transition of phospholipid bilayers

Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993–3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well.By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity.The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Temperature acclimation and phospholipid phase transition in hypothalamic membrane phospholipids of garden lizard, Calotes versicolor

Garden lizards, Calotes versicolor, were acclimated to three different temperatures, i.e., 16°C, 26°C and 36°C for a period of 30 days in ‘walk-in-environmental chambers’. The phospholipid profile and fatty acid pattern were analysed in the hypothalamus and brain of the acclimated animals. Hypothalamic and brain membrane phospholipids were prepared and their phase-transition temperatures were recorded using differential scanning calorimetry. Acclimation temperature, phospholipid composition, fatty acids of these phospholipids and the physical state and fluidity of the specific model membranes of hypothalamus (and brain) are intimately inter-related. Evidence is presented for the first time to show a possible correlation between acclimation temperature and phase-transition temperature of hypothalamic phospholipid membrane. A direct physico-chemical basis is suggested for the thermoregulatory process of hypothalamus leading to a better understanding of our knowledge on the origin of thermoregulation.     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restrictions of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

Phosphatidylcholesterol bilayers. A model for phospholipid-cholesterol interaction

Aqueous dispersions of monovalent and divalent cation salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl) cholesterol form multilamellar vesicles as shown by freeze-fracture electron microscopy, by electron micrographs of the negatively stained liposomes, and by swelling curves of liposomes in hypoosmotic medium. Differential scanning calorimetry reveals that aqueous dispersions of divalent metal salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-cholesterol undergo a characteristic thermotropic phase transition with a relatively large cooperative unit (n > 250 for the calcium salt). In contrast, monovalent cation salts of O-(1,2-dipalmitoyl-sn-glycerol-3-phosphoryl)cholesterol do not show a thermotropic phase transition under comparable conditions. The molecular area of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol in a monolayer is the same in the presence and absence of Ca²⁺, and is virtually equal to the area of an equimolar mixture of dipalmitoyl phosphatidic acid and cholesterol. To account for the novel state induced by Ca²⁺ on aqueous dispersions of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol (i.e., bilayer organization and highly cooperative phase transition), a linear array model is proposed in which Ca²⁺ bridges adjacent arrays of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol molecules, thus freezing the acyl chains in their normal state. One of the main corollaries of the model is that the cooperative unit for a thermotropic phase transition is essentially one-dimensional, rather than a two-dimensional matrix. O-(1,2-Dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol is proposed as an orientationally and conformationally restricted analog of glycerophospholipid plus cholesterol in bilayers.     

A continuum theoretical model and finite elements simulation of bacterial flagellar filament phase transition

The Bacterial flagellar filament can undergo a polymorphic phase transition in response to both mechanical and chemical variations in vitro and in vivo environments. Under mechanical stimuli, such as viscous flow or forces induced by motor rotation, the filament changes its phase from left-handed normal (N) to right-handed semi-coiled (SC) via phase nucleation and growth. Our detailed mechanical analysis of existing experiments shows that both torque and bending moment contribute to the filament phase transition. In this paper, we establish a non-convex and non-local continuum model based on the Ginzburg-Landau theory to describe main characteristics of the filament phase transition such as new-phase nucleation, growth, propagation and the merging of neighboring interfaces. The finite element method (FEM) is adopted to simulate the phase transition under a displacement-controlled loading condition (rotation angle and bending deflection). We show that new-phase nucleation corresponds to the maximum torque and bending moment at the stuck end of the filament. The hysteresis loop in the loading and unloading curves indicates energy dissipation. When the new phase grows and propagates, torque and bending moment remain static. We also find that there is a drop in load when the two interfaces merge, indicating a concomitant reduction in the interfacial energy. Finally, the interface thickness is governed by the coefficients of the gradient of order parameters in the non-local interface energy. Our continuum theory and the finite element method provide a method to study the mechanical behavior of such biomaterials.     

The role of the phospholipid phase transition in the regulation of glucagon binding to lecithin

Glucagon can interact rapidly with multilamellar vesicles of dimyristoyl glycerophosphocholine over a narrow temperature range around or above the phase transition temperature of the pure phospholipid. The temperature dependence of the rates arises, in large part, from glucagon-induced alterations in the phase transition properties of the phospholipid. Similar effects are observed with dilaury glycerophosphocholine but the rate of reaction of glucagon with multilamellar dipalmitoyl glycerophosphocholine is too slow to measure.The rate of reaction of glucagon with equimolar mixtures of two phospholipid molecules has also been studied. Mixtures of dilauryl glycerophosphocholine and distearoyl glycerophosphocholine are known to exhibit lateral phase separation in the gel state. The presence of distearoyl glycerophosphocholine has no effect on the rate of reaction with glucagon, despite the increased number of phase boundaries present. In the case of mixtures of dilauryl glycerophosphocholine and dimyristoyl glycerophosphocholine, glucagon appears to induce some lateral phase separation. This is demonstrated by the ability of glucagon to react rapidly with this lipid mixture, even at temperatures well below the phase transition temperature of the mixture and by differential scanning calorimetry.The thermodynamics of the binding of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine were analyzed with Scatchard plots calculated from measurements of the fluorescence enhancement caused by lipids. Equilibrium binding constants of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine are 1·10⁵ and 5·10⁴ M^?1, respectively. These values are relatively insensitive to temperature, indicating that the equilibrium being measured is between lipid-bound glucagon and free lipid which has had its phase transition properties altered. The number of moles of lipid bound per mole of glucagon decreases markedly above the phase transition temperature. In the water-soluble complex formed between glucagon and dimyristoyl glycerophosphocholine, the peptide binds directly to only 40% of the lipid molecules but, nevertheless, is able to modify the phase transition properties of all of the lipid in the particle.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Molecular dynamics simulations of the interaction of phospholipid bilayers with polycaprolactone

The molecular interaction between common polymer chains and the cell membrane is unknown. Molecular dynamics simulations offer an emerging tool to characterise the nature of the interaction between common degradable polymer chains used in biomedical applications, such as polycaprolactone, and model cell membranes. Herein we characterise with all-atomistic and coarse-grained molecular dynamics simulations the interaction between single polycaprolactone chains of varying chain lengths with a phospholipid membrane. We find that the length of the polymer chain greatly affects the nature of interaction with the membrane, as well as the membrane properties. Furthermore, we next utilise advanced sampling techniques in molecular dynamics to characterise the two-dimensional free energy surface for the interaction of varying polymer chain lengths (short, intermediate, and long) with model cell membranes. We find that the free energy minimum shifts from the membrane-water interface to the hydrophobic core of the phospholipid membrane as a function of chain length. Finally, we perform coarse-grained molecular dynamics simulations of slightly larger membranes with polymers of the same length and characterise the results as compared with all-atomistic molecular dynamics simulations. These results can be used to design polymer chain lengths and chemistries to optimise their interaction with cell membranes at the molecular level.     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

Temperature dependence of the size of phospholipid vesicles

We report measurements of the size of dimyristoyl phosphatidylcholine vesicles over a temperature range that includes the main transition temperature and show that any change in average diameter is less than ±3%.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10–40°C. The fusogenic activity of the cations decreases in the sequence Ca²⁺ > Ba²⁺ > Sr²⁺ Mg²⁺ for cholesterol concentrations in the range 20–40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca²⁺ and Ba²⁺ at 25°C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25°C, Mg²⁺ is ineffective in causing fusion at all cholesterol concentrations. However, at 30°C, Mg²⁺-induced fusion is observed with vesicles containing cholesterol. At 40°C, Mg²⁺ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca²⁺, Ba²⁺ and Sr²⁺. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na⁺ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (T_m) in Na⁺ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the T_m upward, with a sequence of effectiveness Ba²⁺ > Sr²⁺ > Mg²⁺. The T_m of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na⁺, Sr²⁺ or Mg²⁺, the addition of Ba²⁺ reveals endotherms with T_m progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the T_m, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg²⁺ concentrations. With Ca²⁺, saturation is not observed for cholesterol-containing vesicles. The highest rate of fusion for both Ca²⁺- and Mg²⁺-induced fusion is observed with vesicles containing 30 mol% cholesterol.     

A phase transition for a random cluster model on phylogenetic trees

We investigate a simple model that generates random partitions of the leaf set of a tree. Of particular interest is the reconstruction question: what number k of independent samples (partitions) are required to correctly reconstruct the underlying tree (with high probability)? We demonstrate a phase transition for k as a function of the mutation rate, from logarithmic to polynomial dependence on the size of the tree. We also describe a simple polynomial-time tree reconstruction algorithm that applies in the logarithmic region. This model and the associated reconstruction questions are motivated by a Markov model for genomic evolution in molecular biology.     

The kinetics of the main phase transition of aqueous dispersions of phospholipids induced by pressure jump and monitored by raman spectroscopy

The sensitivity of the melting transition temperature of aqueous dispersions of dipalmitoyl- and distearoylphosphatidylcholine to hydrostatic pressure is used to allow measurement of the rates of isothermal freezing and melting of the lipids by rapidly changing the pressure. The degree of order of the lipids is measured by monitoring a ratio of two points in the Raman spectrum of the lipids which changes sharply at the melting temperature. Use of this Raman order ratio allows correlation between the order of the sample and the rates of transition in a manner which is impossible by monitoring only turbidity. Our longest relaxation times range upwards from a few seconds for both compounds. The freezing rates are slowest when the samples are initially fully melted, and the melting rates are slowest when the samples are initially frozen. These results imply that nucleation of the growing phase dominates the kinetics of both freezing and melting.