Chain-melting Transition Research Articles

Phase behavior of two-component lipid membranes: Theory and experiments

The structure of the ripple phase of phospholipid membranes remains poorly understood in spite of a large number of theoretical studies, with many experimentally established structural features of this phase unaccounted for. In this article we present a phenomenological theory of phase transitions in single- and two-component achiral lipid membranes in terms of two coupled order parameters: a scalar order parameter describing lipid chain melting, and a vector order parameter describing the tilt of the hydrocarbon chains below the chain-melting transition. This model reproduces all the salient structural features of the ripple phase, providing a unified description of the phase diagram and microstructure. In addition, it predicts a variant of this phase that does not seem to have been experimentally observed. Using this model we have calculated generic phase diagrams of two-component membranes. We have also determined the phase diagram of a two-component lipid membrane from x-ray diffraction studies on aligned multilayers. This phase diagram is found to be in good agreement with that calculated from the model.

Stalk-free membrane fusion of cationic lipids via an interdigitated phase

We have studied the membrane structure of a cationic lipid (N-t-butyl-N′-tetradecyl-3-tetradecylaminopropionamidine, termed diC14-amidine) using X-ray and neutron small-angle scattering as well as dilatometry and ultrasound velocimetry. At high temperatures we found an anomalously thin lamellar fluid phase, which transforms into a loosely packed interdigitated phase below the chain melting transition. Our finding can be understood based on the specific structure of the lipid backbone, which forms a long spacer between the alkyl chains. The loose lipid packing in the gel phase leads to a significant exposure of the hydrophobic lipid core to the aqueous phase. This reduces energy penalties from the removal of water molecules during fusion and explains the untypically increased fusion properties of amidine in the gel phase. Moreover, this fusion in the gel interdigitated state apparently does not require the formation of stalk intermediate structures.

Temperature and Voltage Dependence of Lipid Membrane Capacitance and the Corresponding Capacitive Currents

Lipid bilayers and monolayers display a voltage dependent chain-melting transition behavior. Such melting transitions also exist in biomembranes close to physiological temperature, and therefore voltage induced transitions may be of biological relevance. Since changes in lipid state involve changes in membrane thickness, one obtains the possibility of a voltage-induced change in capacitance. This phenomenon leads to a capacitance maximum in the melting transition of membranes similar to heat capacity maxima. It further implies, that the often made assumption of constant capacitance in biomembranes (as made in most electrophysiological models) is incorrect close to transitions. As a striking consequence, this finding leads to the possibility of capacitive currents against the applied field with a time-scale of several milliseconds or more, which is the relaxation time of the lipid state after a perturbation by voltage. Simultaneously, the accompanying membrane permeability changes resulting in an ion current along the applied field.

A kinetic lattice model of liquid-to-gel phase transition in lipid membrane: Experimental calibration and physiological implications

A kinetic lattice model of chain-melting phase transition has been developed. Its time scale has been calibrated with the acoustic relaxation spectroscopic data. The model permits analysis and predictions of kinetic features of those physiological processes in biological membranes the mechanism of which is based in a phase transition in the lipid component. It is shown that the phase-transition mechanism can sustain the high rate of synaptic exocytosis known for the fastest synapses in the central nervous system. It is also demonstrated that this mechanism can be an essential factor of synaptic plasticity.

Phase Transitions and Spatially Ordered Counterion Association in Ionic-Lipid Membranes: Theory versus Experiment

Aqueous dispersions of phosphatidylglycerol (PG) lipids may present an anomalous chain-melting transition at low ionic strengths, as seen by different experimental techniques such as calorimetry or light scattering. The anomaly disappears at high ionic strengths or for longer acyl-chain lengths. In this article, we use a statistical model for the bilayer that distinguishes both lipid chain and headgroup states in order to compare model and experimental thermotropic and electrical properties. The effective van der Waals interactions among hydrophobic chains compete with the electrostatic repulsions between polar headgroups, which may be ionized (counterion dissociated) or electrically neutral (associated with counterions). Electric degrees of freedom introduce new thermotropic charge-ordered phases in which headgroup charges may be spatially ordered, depending on the electrolyte ionic strength, introducing a new rationale for experimental data on PGs. The thermal phases presented by the model for different chain lengths, at fixed ionic strength, compare well with an experimental phase diagram constructed on the basis of differential scanning calorimetry profiles. In the case of dispersions of DMPG (dimyristoyl phosphatidylglycerol) with added monovalent salt, the model properties reproduce the main features displayed by data from differential scanning calorimetry as well as the characteristic profile for the degree of ionization of the bilayer surface across the anomalous transition region, obtained from the theoretical interpretation of electrokinetic (conductivity and electrophoretic mobility) measurements.

Effect of Increase in Orientational Order of Lipid Chains and Head Group Spacing on Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion

Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs), have been shown by us to induce fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (<10 mol %) at temperatures above the chain-melting transition. Low concentration cholesterol (<10 mol %), above the gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration cholesterol on the fusion induced by the three drugs, which has been explained by parsing the effect of orientational order and increase in head group spacing on the fusion process.

Multiphoton Polarization Imaging of Steady-State Molecular Order in Ternary Lipid Vesicles for the Purpose of Lipid Phase Assignment

We have investigated lipid acyl chain order parameters of giant unilamellar vesicles (GUVs) using multiphoton fluorescence microscopy. We compare two widely used models of lipid acyl chain order parameters: the "wobble-on-a-cone" model and the Gaussian distribution model. For the first time, we systematically address a ternary system for which the phase diagram encompassing both composition and temperature space has been mapped in order to determine tie-line directions and thus phase assignment. In addition, because miscibility and chain melting transitions can be observed directly and simultaneously with multiphoton microscopy, our technique is applicable to determining the extent of the coupling between chain order and miscibility; thus, it provides a more robust platform for comparison with theory.

Pore Formation in a Binary Giant Vesicle Induced by Cone-Shaped Lipids

We have investigated shape deformations of binary giant unilamellar vesicles (GUVs) composed of cone- and cylinder-shaped lipids. By coupling the spontaneous curvature of lipids with the phase separation, we demonstrated pore opening and closing in GUVs. When the temperature was set below the chain melting transition temperature of the cylinder-shaped lipid, the GUVs burst and then formed a single large pore, where the cone shape lipids form a cap at the edge of the bilayer to stabilize the pore. The pore closed when we increased the temperature above the transition temperature. The pore showed three types of shapes depending on the cone-shaped lipid concentration: simple circular, rolled-rim, and wrinkled-rim pores. These pore shape changes indicate that the distribution of the cone- and cylinder-shaped lipids is asymmetric between the inner and outer leaflets in the bilayer. We have proposed a theoretical model for a two-component membrane with an edge of bilayer where lipids can transfer between two leaflets. Using this model, we have reproduced numerically the observed shape deformations at the rim of pore.

Packing behaviour of two predominant anionic phospholipids of bacterial cytoplasmic membranes

Phosphatidylglycerol and cardiolipin represent the most abundant anionic phospholipid components of cytoplasmic bacterial membranes and thus are used as constituents for membrane mimetic systems. In this study, we have characterized the temperature dependent phase behaviour of the binary system dipalmitoyl-phosphatidylglycerol (DPPG) and tetramyristoyl-cardiolipin (TMCL) using microcalorimetry and X-ray scattering techniques. Both lipids exhibited a very similar main transition temperature (∼41°C), showing a minimum (39.4°C) for the binary mixtures at XDPPG=0.8, and exhibited low-temperature phase transitions, which were abolished by incorporation of small amounts (≤10mol%) of the other lipid component. Therefore, over a wide temperature and composition range a lamellar Lβ gel phase is the predominant structure below the chain melting transition, characterized by a relatively broad wide-angle peak for XDPPG≤0.8. This observation suggests the existence of packing inconsistencies of the TMCL/DPPG hydrocarbon lattices in the gel phase, supported by the small average size of lipid clusters (∼50 lipids) within this composition range. The bilayer thickness for the lamellar-gel phase showed a monotonic increase (56Å for TMCL to about 58Å for XDPPG=0.8 at 30°C), which may be explained by different degrees of partial interdigitation of the acyl chains to compensate for the differences in the hydrocarbon lengths of DPPG and TMCL in the Lβ phase.

RNA and DNA interactions with zwitterionic and charged lipid membranes — A DSC and QCM-D study

The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na(+)) or divalent (Ca(2+)) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC-DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC-DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA-phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine.

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High-density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic alpha-helices that have high content (> or = 1/3) and distinct distribution of charged and apolar residues, adopt molten globule-like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA-I, shows relatively large volume contraction, DeltaV(unf) = -0.33%, and an apparent reduction in thermal expansivity upon unfolding, Deltaalpha(unf) < or = 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon alpha-helical unfolding, which may result from disruption of multiple salt bridges. At 5 degrees C, apoA-I shows large thermal expansion coefficient, alpha(5 degrees) = 15.10(-4) K(-1), that rapidly declines upon heating from 5 to 40 degrees C, alpha(40 degrees) - alpha(5 degrees) = -4.10(-4) K(-1); apolipoprotein C-I shows similar values of alpha(5 degrees) and alpha(40 degrees). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein-lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA-I or apoC-I, dimyristoyl phosphatidylcholine, and 0-20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport.

Effect of Trans Unsaturation on Molecular Organization in a Phospholipid Membrane

We employed solid state (2)H NMR, complemented by computer simulations, to compare molecular organization in model membranes composed of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC), 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC), and 1,2-distearoylphosphatidylcholine (18:0-18:0PC). These phospholipids have elaidic acid (EA) containing a trans double bond, oleic acid (OA) containing a cis double bond, and saturated stearic acid (SA), respectively, at the sn-1 position and were synthesized with perdeuterated SA at the sn-2 position. The temperature of the chain melting transition is depressed less for t18:1-18:0PC (31.5 degrees C) than c18:1-18:0PC (7 degrees C) relative to 18:0-18:0PC (53 degrees C), reflecting the smaller deviation from the linear conformation produced by a trans as opposed to cis double bond. Acyl chain order in t18:1-18:0PC (S(CD) = 0.135) in the liquid crystalline state is much closer to that of c18:1-18:0PC (S(CD) = 0.128) than that of the substantially more ordered 18:0-18:0PC (S(CD) > 0.156), which is attributed to the reduced energy barrier to rotation about the C-C single bonds next to either a trans or cis carbon double bond. A conformation that somewhat resembles a saturated chain and an intrinsic disorder approaching that of a cis unsaturated chain characterize EA and, we speculate, may play a role in the adverse impact dietary trans fatty acids (TFA) have on biological function.

Structural and thermodynamic determinants of chain-melting transition temperatures for phospholipid and glycolipids membranes

For optimum function, biological membranes need a fluid environment, which is afforded by the liquid-disordered phase of lipids with low chain-melting temperatures or the liquid-ordered phase that is formed by combining high chain-melting lipids with cholesterol. The dependence of chain-melting transition temperature on lipid chain structure is therefore of central importance. The currently available database, including sphingolipids and glycolipids, is summarised here by parameterising systematic dependences on molecular structure in terms of suitable thermodynamic models. Chain-length dependence, chain asymmetry of lipids forming partially interdigitated and mixed interdigitated gel phases, chain unsaturation, positional dependence of methyl branching, headgroup-attached and α-branched chains, and length of zwitterionic headgroups are all covered. This type of information is essential for biophysical approaches to functional lipidomics.

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.

The Temperature Dependence And Quantized Nature Of The Lipid Membrane Permeability

We investigate the permeability of phospholipid membranes for fluorescence dyes and ions. We find that the permeability is strongly enhanced close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence Correlation Spectroscopy (FCS) reveals the permeability for rhodamine dyes across 100 nm vesicles. Using FCS, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events. Furthermore, we demonstrate that general anaesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anaesthetics decrease channel conductance and ultimately lead to ‘blocking’ of the lipid pores in experiments performed at or above the chain melting transition. Our data suggests that the macroscopic increase in permeability close to transitions and microscopic lipid channel formation are the same physical process.

The Effect of Trans Unsaturation on Molecular Organization in a Phospholipid Membrane

We employed solid state 2H NMR, complemented by computer simulations, to compare molecular organization in model membranes composed of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC), 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC), and 1,2-distearoylphosphatidylcholine (18:0-18:0PC). These phospholipids have elaidic acid (EA) containing a trans double bond, oleic acid (OA) containing a cis double bond, and saturated stearic acid (SA), respectively, at the sn-1 position and were synthesized with perdeuterated SA at the sn-2 position. The temperature of the chain melting transition is depressed less for t18:1-18:0PC (31.5 °C) than c18:1-18:0PC (7 °C) relative to 18:0-18:0PC (53 °C), reflecting the smaller deviation from the linear conformation produced by a trans as opposed to cis double bond. Acyl chain order in t18:1-18:0PC (SCD = 0.135) in the liquid crystalline state is much closer to that of c18:1-18:0PC (SCD = 0.128) than that of the substantially more ordered 18:0-18:0PC (SCD > 0.15...

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...

Pressure Perturbation Calorimetric Studies on Phospholipid-Sterol Mixtures

Sterols regulate biological processes and sustain the lateral structure of cellular membranes. The sterol cholesterol, its precursor lanosterol, the plant sterols stigmasterol and ergosterol as well as 7-dehydrocholesterol were added up to 36 mol-% to vesicles of the phospholipid 1,2-dipalmitoyl-snglycero- 3-phosphatidylcholine (DPPC). The aim of this study was to investigate the influence of the sterol side chain and ring structure on the volumetric properties of the lipid bilayer system by using pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the thermal expansion coefficient and volumetric properties of biomolecules. The experiments were carried out in the temperature range from 10 to 85 °C, i. e., at temperatures below and above the chain-melting transition temperatures of the lipid mixtures. Additionally, corresponding differential scanning calorimetric (DSC) measurements were carried out.Whereas the conformational properties of the different sterols have a significant effect on the order parameter of the lipid acyl-chains, the thermodynamic parameters of these sterols are less influenced by the differential structural changes of the sterols. For lanosterol and stigmasterol, marked differences are found, however.

Energetics of Hydrophobic Matching in Lipid-Protein Interactions

Lipid chain length modulates the activity of transmembrane proteins by mismatch between the hydrophobic span of the protein and that of the lipid membrane. Relative binding affinities of lipids with different chain lengths are used to estimate the excess free energy of lipid-protein interaction that arises from hydrophobic mismatch. For a wide range of integral proteins and peptides, the energy cost is much less than the elastic penalty of fully stretching or compressing the lipid chains to achieve complete hydrophobic matching. The chain length dependences of the free energies of lipid association are described by a model that combines elastic chain extension with a free energy term that depends linearly on the extent of residual mismatch. The excess free energy densities involved lie in the region of 0.5–2.0kBT.nm−2. Values of this size could arise from exposure of hydrophobic groups to polar portions of the lipid or protein, but not directly to water, or alternatively from changes in tilt of the transmembrane helices that are energetically comparable to those activating mechanosensitive channels. The influence of hydrophobic mismatch on dimerization of transmembrane helices and their transfer between lipid vesicles, and on shifts in chain-melting transitions of lipid bilayers by incorporated proteins, is analyzed by using the same thermodynamic model. Segmental order parameters confirm that elastic lipid chain distortions are insufficient to compensate fully for the mismatch, but the dependence on chain length with tryptophan-anchored peptides requires that the free energy density of hydrophobic mismatch should increase with increasing extent of mismatch.