Phase Transitions In Lipid Membranes Research Articles

Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water.

Morphological and gel-to-liquid phase transitions of lipid membranes are generally considered to primarily depend on the structural motifs in the hydrophobic core of the bilayer. Structural changes in the aqueous headgroup phase are typically not considered, primarily because they are difficult to quantify. Here, we investigate structural changes of the hydration shells around large unilamellar vesicles (LUVs) in aqueous solution, using differential scanning calorimetry (DSC), and temperature-dependent ζ-potential and high-throughput angle-resolved second harmonic scattering measurements (AR-SHS). Varying the lipid composition from 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) to 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), to 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS), we observe surprisingly distinct behavior for the different systems that depend on the chemical composition of the hydrated headgroups. These differences involve changes in hydration following temperature-induced counterion redistribution, or changes in hydration following headgroup reorientation and Stern layer compression.

Transient Permeabilization of Living Cells: Combining Shear Flow and Acoustofluidic Trapping for the Facilitated Uptake of Molecules

Here, we present a novel approach for the transient permeabilization of cells. We combined laminar shear flow in a microchannel with chaotic advection employing surface acoustic waves. First, as a fundamental result on the one hand, and as a kind of reference measurement for the more complex acoustofluidic approach on the other hand, we studied the permeabilization of cells in pure shear flow in a microchannel with Y-geometry. As a proof of principle, we used fluorescent dyes as model drugs and investigated their internalization into HeLa cells. We found that drug uptake scaled non-linearly with flow rate and thus shear stress. For calcein, we obtained a maximal enhancement factor of about 12 at an optimum flow rate of Q = 500 µL/h in the geometry used here compared to static incubation. This result is discussed in the light of structural phase transitions of lipid membranes accompanied by non-linear effects, as the plasma membrane is the main barrier to overcome. Second, we demonstrated the enhanced permeabilization of acoustically trapped cells in surface acoustic wave induced vortices in a microchannel, with an enhancement factor of about 18 compared to quasi-static incubation. Moreover, we optimized the trapping conditions regarding flow rate, the power level of the surface acoustic wave, and trapping time. Finally, we showed that our method is not limited to small molecules but can also be applied to compounds with higher molecular weight.

Effect of TMAO on the Structure and Phase Transition of Lipid Membranes: Potential Role of TMAO in Stabilizing Cell Membranes under Osmotic Stress.

Extremophiles adopt strategies to deal with different environmental stresses, some of which are severely damaging to their cell membrane. To combat high osmotic stress, deep-sea organisms synthesize osmolytes, small polar organic molecules, like trimethylamine-N-oxide (TMAO), and incorporate them in the cell. TMAO is known to protect cells from high osmotic or hydrostatic pressure. Several experimental and simulation studies have revealed the roles of such osmolytes on stabilizing proteins. In contrast, the effect of osmolytes on the lipid membrane is poorly understood and broadly debated. A recent experiment has found strong evidence of the possible role of TMAO in stabilizing lipid membranes. Using the molecular dynamics (MD) simulation technique, we have demonstrated the effect of TMAO on two saturated fully hydrated lipid membranes in their fluid and gel phases. We have captured the impact of TMAO's concentration on the membrane's structural properties along with the fluid/gel phase transition temperatures. On increasing the concentration of TMAO, we see a substantial increase in the packing density of the membrane (estimated by area, thickness, and volume) and enhancement in the orientational order of lipid molecules. Having repulsive interaction with the lipid head group, the TMAO molecules are expelled away from the membrane surface, which induces dehydration of the lipid head groups, increasing the packing density. The addition of TMAO also increases the fluid/gel phase transition temperature of the membrane. All of these results are in close agreement with the experimental observations. This study, therefore, provides a molecular-level understanding of how TMAO can influence the cell membrane of deep-sea organisms and help in combating the osmotic stress condition.

Dember photovoltaic effect as method for structural characterization of phospholipidic membranes

Dember photovoltaic effect (Dember effect) was used to study permeability of a thin layer of lyotropic liquid crystal (dipalmitoylphosphatidylcholine) at various temperatures. By photo dissociation of a photosensitive ionic dye dissolved in a lyotropic liquid crystal, an inhomogeneous distribution of charge carriers was induced in a thin liquid crystal layer. The diffusion relaxation of these charge distributions depends on the structure of lyotropic liquid crystal and show up itself in the temperature dependence of pulsed photoelectric response. The data obtained are in a good agreement with the results of dielectric measurements. In perspective, above method can be applied to examine permeability of natural and synthetic lipid vesicles (cells, exosomes, liposomes and other vesicles) relevant in medicine and to characterize the structural phase transitions in lipid membranes when modified by molecules of interest (e.g. cholesterol in liposomes to increase rigidity and stability).

On the Origin of the Anomalous Behavior of Lipid Membrane Properties in the Vicinity of the Chain-Melting Phase Transition

Biomembranes are key objects of numerous studies in biology and biophysics of great importance to medicine. A few nanometers thin quasi two-dimensional liquid crystalline membranes with bending rigidity of a few kT exhibit unusual properties and they are the focus of theoretical and experimental physics. The first order chain-melting phase transition of lipid membranes is observed to be accompanied by a pseudocritical behavior of membrane physical-chemical properties. However, the investigation of the nature of the anomalous swelling of a stack of lipid membranes in the vicinity of the transition by different groups led to conflicting conclusions about the level of critical density fluctuations and their impact on the membrane softening. Correspondingly, conclusions about the contribution of Helfrich’s undulations to the effect of swelling were different. In our work we present a comprehensive complementary neutron small-angle and spin-echo study directly showing the presence of significant critical fluctuations in the vicinity of the transition which induce membrane softening. However, contrary to the existing paradigm, we demonstrate that the increased undulation forces cannot explain the anomalous swelling. We suggest that the observed effect is instead determined by the dominating increase of short-range entropic repulsion.

Amphiphilic copolymers change the nature of the ordered-to-disordered phase transition of lipid membranes from discontinuous to continuous.

The phase behaviour as a function of temperature is explored for pure phospholipid (DPPC) and hybrid lipid-polymer (DPPC/Pluronic L64) bilayers with the aid of atomistic MD simulations. The range of the fixed-temperature simulations includes temperatures below and above the known melting temperature (Tm) for DPPC membranes. For the pure lipid bilayer, the main phase transition is discontinuous, as verified by the abrupt changes observed in the membrane structure, elasticity and the lipid diffusivity near the critical temperature Tm, which lies in the region 298.15-303.15 K. A pre-transition step is detected at 298.15 K which has been identified as the ripple phase (Pβ'), where ordered and disordered lipids coexist, causing thickness fluctuations. In the ordered gel phase, the positional ordering as assessed by the lipid radial distribution functions is long-range and some degree of hexagonal packing is measured. The hybrid bilayers on the other hand, transform from a more ordered to a disordered phase in a continuous manner, without finite jumps in their properties. No signs of the ripple phase are identified and the ordered phase exhibits very limited hexagonal packing and some positional ordering that decays fast. The effect of the inserted polymers in the two phases is reversed; at low temperatures, they render the membrane thinner, less cohesive and less ordered compared to the pure one, with the lipids assuming faster diffusion rates, whereas at high temperatures, the polymer interaction with the lipids acts reducing their diffusivity, but also increasing the lipid tail ordering and the membrane stiffness. The ability of the amphiphilic L64 copolymers to change the nature of the main phase transition of lipid membranes and their properties both in the ordered and the disordered phase is of vital importance for the prediction of both the efficacy of hybrid lipid/polymer nanoparticles as drug delivery vehicles as well as their potential adverse implications during interactions with healthy cell membranes.

Temperature-Dependent Lipid Extraction from Membranes by Boron Nitride Nanosheets.

Two-dimensional (2D) materials can mechanically insert into cell membranes and extract lipids out, thus leading to the destruction of cell integrity. On the one hand, the cytotoxicity of 2D materials can be harnessed in surface engineering to resist biofouling, and on the other hand, it causes great concern with in vivo biomedical applications ranging from drug delivery to nanoimaging. To understand the nature of this cytotoxic behavior and find strategies to control it, we performed molecular dynamics (MD) simulations on the lipid extraction of hexagonal boron nitride (BN) nanosheets from lipid membranes. Interestingly, we observed that the lipid extraction behavior suddenly disappears as temperature decreases. Structural analyses revealed that this temperature dependence is related to the lipid membrane phase transition, which was confirmed by an additional membrane model with phase state regulated by cholesterol. The potential of mean force calculation was adopted to clarify the thermodynamic origin of these results, which also indicates directions to adjust the lipid extraction behavior of nanomaterials. Overall, this work suggests that the cytotoxic mechanical interactions between 2D materials and cell membranes can be controlled by temperature and other factors which can induce phase transitions of lipid membranes and that the thermodynamic threshold of the lipid extraction varies for surfaces with different curvature. This work clarifies the thermodynamics in the lipid extraction phenomenon of 2D materials and indicates possible strategies to adjust the antibacterial performance or cytotoxicity of 2D materials.

Low-pH-Induced Lamellar to Bicontinuous Primitive Cubic Phase Transition in Dioleoylphosphatidylserine/Monoolein Membranes.

Electrostatic interactions (EIs) play important roles in the structure and stability of inverse bicontinuous cubic (QII) phases of lipid membranes. We examined the effect of pH on the phase of dioleoylphosphatidylserine (DOPS)/monoolein (MO) membranes at low ionic strengths using small-angle X-ray scattering (SAXS). We found that the phase transitions from lamellar liquid-crystalline (Lα) to primitive cubic (QIIP) phases in DOPS/MO (2/8 molar ratio) membranes occurred in buffers containing 50 mM NaCl at and below the final pH of 2.75 as the pH of the membrane suspension was decreased from a neutral value. The kinetic pathway of this transition was revealed using time-resolved SAXS with a stopped-flow apparatus. The first step is a rapid transition from the Lα phase to the hexagonal II (HII) phase, and the second step is a slow transition from the HII phase to the QIIP phase. We determined the rate constants of the first step, k1, and of the second step, k2, by analyzing the time course of SAXS intensities quantitatively. The k1 value increased with temperature. The analysis of this result provided the values of its apparent activation energy, which were constant over temperature but increased with pH. This can be explained by an EI effect on the free energy of the transition state. In contrast, the k2 value decreased with temperature, indicating that the true activation energy increased with temperature. These experimental results were analyzed using the theory of the activation energy of phase transitions of lipid membranes when the free energy of the transition state depends on temperature. On the basis of these results, we discussed the mechanism of this phase transition.

Activation Energy of the Low-pH-Induced Lamellar to Bicontinuous Cubic Phase Transition in Dioleoylphosphatidylserine/Monoolein.

Electrostatic interaction is an important factor for phase transitions between lamellar liquid-crystalline (Lα) and inverse bicontinuous cubic (QII) phases. We investigated the effect of temperature on the low-pH-induced Lα to double-diamond cubic (QII(D)) phase transition in dioleoylphosphatidylserine (DOPS)/monoolein (MO) using time-resolved small-angle X-ray scattering with a stopped-flow apparatus. Under all conditions of temperature and pH, the Lα phase was directly transformed into an intermediate inverse hexagonal (HII) phase, and subsequently the HII phase slowly converted to the QII(D) phase. We obtained the rate constants of the initial step (i.e., the Lα to HII phase transition) and of the second step (i.e., the HII to QII(D) phase transition) using the non-negative matrix factorization method. The rate constant of the initial step increased with temperature. By analyzing this result, we obtained the values of its apparent activation energy, Ea (Lα → HII), which did not change with temperature but increased with an increase in pH. In contrast, the rate constant of the second step decreased with temperature at pH 2.6, although it increased with temperature at pH 2.7 and 2.8. These results indicate that the value of Ea (HII → QII(D)) at pH 2.6 increased with temperature, but the values of Ea (HII → QII(D)) at pH 2.7 and 2.8 were constant with temperature. The values of Ea (HII → QII(D)) were smaller than those of Ea (Lα → HII) at the same pH. We analyzed these results using a modified quantitative theory on the activation energy of phase transitions of lipid membranes proposed initially by Squires et al. (Squires, A. M.; Conn, C. E.; Seddon, J. M.; Templer, R. H. Soft Matter 2009, 5, 4773). On the basis of these results, we discuss the mechanism of this phase transition.

Fluctuations of systems in finite heat reservoirs with applications to phase transitions in lipid membranes

In an adiabatically shielded system, the total enthalpy is conserved. Enthalpy fluctuations of an arbitrarily chosen subsystem must be buffered by the remainder of the total system which serves as a heat reservoir. The magnitude of these fluctuations depends on the size of the reservoir. This leads to various interesting consequences for the physical behavior of the subsystem. As an example, we treat a lipid membrane with a phase transition that is embedded in an aqueous reservoir. We find that large fluctuations are attenuated when the reservoir has finite size. This has consequences for the compressibility of the membrane since volume and area fluctuations are also attenuated. We compare the equilibrium fluctuations of subsystems in finite reservoirs with those in periodically driven systems. In such systems, the subsystem has only finite time available to exchange heat with the surrounding medium. A larger frequency therefore reduces the volume of the accessible heat reservoir. Consequently, the fluctuations of the subsystem display a frequency dependence. While this work is of particular interest for a subsystem displaying a transition such as a lipid membrane, some of the results are of a generic nature and may contribute to a better understanding of relaxation processes in general.

Precise and millidegree stable temperature control for fluorescence imaging: application to phase transitions in lipid membranes.

We present the design of a custom temperature-controlled chamber suitable for water or oil immersion fluorescence microscopy and its application to phase behavior in lipid bilayer vesicles. The apparatus is self-contained and portable, suitable for multiuser microscopy facilities. It offers a higher temperature resolution and stability than any comparable commercial apparatus, on the order of millidegrees. We demonstrate the utility of the system in the study of miscibility transitions in model membranes. The temperature-dependent phase behavior of model membrane systems that display liquid-ordered (L(o)) phase coexistence with the liquid-disordered (L(d)) phase is relevant to understanding the existence of heterogeneities in biological cell plasma membranes, ubiquitously termed "lipid rafts."

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140–9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid–crystalline membranes.

Transient Ordered Domains in Single-Component Phospholipid Bilayers

We report evidence of dense, ordered nanodomains in single-component fluid lipid bilayers. Our atomic-scale molecular dynamics simulations suggest that the area available to a lipid acyl chain exhibits large fluctuations, resulting in denser and sparser domains. The sizes of the dense domains can be up to approximately 10 nm, and their lifetimes are of the order of approximately 10 ns. In addition, our simulations suggest that domains of lipids with highly ordered acyl chains form predominantly within the dense regions, their sizes ranging from a few chains up to a few nanometers, and with lifetimes between approximately 10 ps-10 ns. These observations shed light on the origin of experimentally observed fluctuations, as well as on the mechanisms of phase transitions in lipid membranes.

膜の張力変化が誘起する脂質膜の相転移

膜の張力変化が誘起する脂質膜の相転移

Transbilayer Movement of Phospholipids at the Main Phase Transition of Lipid Membranes: Implications for Rapid Flip-Flop in Biological Membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C 6-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC ( T c = 41°C) and DMPC ( T c = 23°C). The flip-flop of analogs was fast at the T c of DPPC with a half-time ( t 1/2) of ∼6–10 min and even faster at the T c of DMPC with t 1/2 on the order of <2 min, as shown for C 6-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.

A Theoretical Analysis of Intracellular Oxygen Diffusion

Oxygen diffusion rates within cells may be heterogeneous, with more rapid diffusion occurring along intracellular pathways of high oxygen solubility, such as mitochondria. Recent experimental data indicate that tissue oxygen permeability rises sharply in a temperature range associated with phase transitions in lipid membranes suggesting that membranes may function as oxygen “pathway”. The experimental data have been analyzed using theoretical models of diffusion in two-phase media. By assuming muscles to be composed entirely of aqueous cytosol and lipids, cytosolic permeability was determined as a function of temperature by matching the experimental values of tissue permeability with those of model predictions usingin vitrovalues of lipid permeability. Cytosolic permeability ranged from 50% of water permeability (low temperature) to 90% of water permeability (high temperature) and its temperature dependence was distinctly different from that of water. An upper bound for cytosolic permeability was calculated using a physiologic value for protein volume fraction, and lipid permeability was obtained using this cytosolic permeability. A model with a parallel arrangement of lipid and cytosol yielded a value of lipid permeability that was 71% higher than thein vitrovalue. Intracellular permeabilities calculated from tissue permeability values were found to be inconsistent with experimentally reported values for rat cardiac myocytes. Comparison of three different muscles exhibited the same trend of higher permeability with higher lipid content. It is concluded that both lipid and cytosol permeabilities and, hence, tissue permeabilities are different among different muscles and one should exercise caution when data from one muscle is used to calculate or extrapolate values in other muscles. It is conceivable that muscles with very high mitochondrial content, such as diaphragm and cardiac muscles may exhibit an oxygen permeability which is significantly higher than commonly accepted values. These results warrant additional measurements of tissue oxygen permeability at 37 °C, especially for oxidative muscles with high lipid content.

Ultrasonic studies of phase transitions in lipid membranes and protein molecules

This paper reviews phase transitions in the lipid membranes and protein molecules studied by means of ultrasonic (sound velocity and absorption) and related methods (calorimetric, volumetric, and others). Peculiarities of phase transitions in small-dimension systems in an aqueous environment (a finite width of the transition zone, probability of critical nuclei formation, hysteresis, hydration changes, etc.) are discussed. A comparison of ultrasonic and calorimetric methods showing their complementarity in the studies of kinetics of phase transitions is presented. Ultrasonic data on the melting of lipid vesicles in aqueous dispersions are reviewed. Recent achievements in the ultrasonic study of the kinetic mechanism of creation and growth of gel nuclei within the parent liquid-crystalline phase are reported. Ultrasonic studies of the protein/lipid interactions are reviewed and perspectives are outlined. Native to molten globule state transition in globular proteins is considered (volume and compressibility changes, acoustic relaxation, protein interior hydration). The differences in the behavior of the phase transitions in membranes and in protein molecules are discussed. New questions for further ultrasonic studies of the phase transitions are formulated.

リン脂質の指組み構造膜

The ability of bilayer lipids to form interdigitated states, in which the acyl chains from opposing monolayers interdigitate, has recently come into focus as a means to significantly alter membrane properties. In addition to changing the membrane thickness and the hydrophobic region of bilayer membranes, the formation of interdigitated membranes alters the electrostatic properties of the membrane surface. A variety of molecules can induce the formation of a fully interdigitated phase by saturated symmetric phospholipids. They include amphipathic molecules such as polyols, short chain alcohols, anesthetics and so on. Common properties are that they do not penetrate too deeply into the hydrocarbon core of bilayer membranes and that they are capable of displacing some of the water molecules associated with the head groups of lipids, thereby creating an increase in the surface area of these groups.In the present review, the forces governing the self-assembly of lipid molecules into interdigitated structures, the physical properties distinctive of interdigitated state membranes and fruitful methods by which interdigitation in membranes may be determined and characterized are outlined along with a detection method developed by the author and coworkers. Mechanisms of phase transition of lipid membranes in the presence of ethanol as an inducer are also proposed and covered together with the results of research concerning the effects of cholesterol on the formation of interdigitated membranes. The possibility of producing formation of interdigitated structures in biomembranes is discussed.

Molecular mechanism of inhalation anesthesia and phase transition of lipid membranes

Abstract This paper is dedicated to Professor Mase, S. of Kyushu University. In this paper the molecular mechanism of inhalation anesthesia is reviewed. The effect of anesthetics upon the phase transition temperature of lipid membranes correlates with the anesthetic potency. Hydrostatic pressure antagonizes the anesthetic effect. Thus, inhalation anesthesia is considered to be a physico-chemical process. The transition temperature of lipid membranes is colligatively depressed by anesthetics. On the other hand, certain anesthetics broaden the width of the temperature span of the transition, while others do not. Solid solution theory is employed to elucidate the depression of the phase transition temperature and the width of the temperature span of the transition simultaneously.

Non-linear characteristics of lipid monolayers in relation to the ability to self-organize

It is known that electrostatic interactions strongly influence the growth of condensed phase domains at the main phase transition of lipid membranes. It is demonstrated in this paper that the inclusion of electrostatic interactions into the pressure-area-temperature equation of state provides a much better fit to the observed equilibrium behaviour than the classical van der Waals form. Both of these equations of state predict a region of negative differential compressibility (NDC), which is not described on long time scales because of a competing first-order phase transition. It is shown in this paper that dynamic NDC can account for most of the features of the observed oscillatory behaviour of monolayers at the oil-water interface. Such behaviour may be related to self-organization of spatiotemporal structures in living organisms.