Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.     

Partition of amphiphilic molecules to lipid bilayers by isothermal titration calorimetry

The partition of the amphiphile sodium dodecyl sulfate (SDS) between an aqueous solution and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was followed by isothermal titration calorimetry (ITC) as a function of the total concentration of SDS. It was found that the obtained partition coefficient is strongly affected by the ligand concentration, even after correction for the charge imposed in the bilayer by the bound SDS. The partition coefficient decreased as the total concentration of SDS increased, with this effect being significant for local concentrations of SDS in the lipid bilayer above 5 molar%. At those high local concentrations, the properties of the lipid bilayer are strongly affected, leading to nonideal behavior and concentration-dependent apparent partition coefficients. It is shown that with the modern ITC instruments available, the concentrations of SDS can be drastically reduced while maintaining a good signal-to-noise ratio. The intrinsic parameters of the interaction with unperturbed membranes can be obtained from the asymptotic behavior of the apparent parameters as a function of the ligand concentration for both nonionic and ionic solutes. A detailed analysis is performed, and a spreadsheet is provided to obtain the interaction parameters with and without correction for electrostatics.     

Applications of pressure perturbation calorimetry in biophysical studies

Pressure perturbation calorimetry (PPC) is a relatively new and efficient technique, to study the volumetric properties of biomolecules in solution. In PPC, the coefficient of thermal expansion of the partial volume of the biomolecule is deduced from the heat consumed or produced after small isothermal pressure jumps (typically ± 5 bar). This strongly depends on the interaction of the biomolecule with the solvent or cosolvent as well as on its packing and internal dynamic properties. This technique, complemented by ultrasound velocity and densitometry, provides valuable insight into the basic thermodynamic properties of solvation and volume effects accompanying phase transitions and interactions of biomolecular systems. Here we review data on protein folding, ligand binding processes, and phospholipid phase transitions, together with discussion of interpretation and further significant applications.     

The hydration pressure between lipid bilayers. Comparison of measurements using x-ray diffraction and calorimetry.

The hydration pressure between dipalmitoyl phosphatidyl-N,N-dimethylethanolamine (DPPE-Me2) bilayers has been analyzed by both x-ray diffraction measurements of osmotically stressed liposomes and by differential scanning calorimetry. By the x-ray method, we obtain a magnitude (Po) and decay length (lambda) for the hydration pressure which are both quite similar to those found for bilayers of other zwitterionic lipids, such as phosphatidylcholines. That is, x-ray analysis of DPPE-Me2 in the gel phase gives lambda = 1.3 A, the same as that previously measured for the analogous gel phase lipid dipalmitoylphosphatidylcholine (DPPC), and Po = 3.9 x 10(9) dyn/cm2, which is in excellent agreement with the value of 3.6 x 10(9) dyn/cm2 calculated from the measured Volta potential of DPPE-Me2 monolayers in equilibrium with liposomes. These results indicate that the removal of one methyl group to convert DPPC to DPPE-Me2 does not markedly alter the range or magnitude of the hydration pressure. Calorimetry shows that the main gel to liquid-crystalline phase transition temperature of DPPE-Me2 is approximately constant for water contents ranging from 80 to 10 water molecules per lipid molecule, but increases monotonically with decreasing water content below 10 waters per lipid. A theoretical fit to these temperature vs. water content data predicts lambda = 6.7 A. The difference in observed values of lambda for x-ray and calorimetry measurements can be explained by effects on the thermograms of additional intra- and intermolecular interactions which occur at low water contents where apposing bilayers are in contact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors determining pressure perturbation calorimetry measurements: evidence for the formation of metastable states at lipid phase transitions

The factors that influence the application of pressure perturbation calorimetry in studying the volume change of the phase transition of lipids are discussed. These factors include a correction for the temperature-shift induced by perturbation, the kinetic irreversibility of the phase transition and the magnitude of the pressure perturbation. We take into account the fact that the dependence of the phase transition temperature on pressure will affect the temperature-shift induced by pressure. As a result, there is a discrepancy between the compression part of the cycle and the expansion. In addition, sequential cycles lead to a gradual loss in magnitude of the heat effect upon pressure perturbation. We suggest that these phenomena can be explained by the formation of a metastable glass-like state that converts to a stable phase at temperatures removed from the region of the phase transition.     

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding

BackgroundPressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules.Scope of this reviewThis review provides novel details of the use of PPC to analyze unfolding transitions in proteins. The emphasis is made on the data analysis as well as on the validation of different structural factors that define the volume changes upon unfolding. Four case studies are presented that show the application of these concepts to various protein systems.Major conclusionsThe major conclusions are:

• 1.Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles.
• 2.The changes in the thermal expansion coefficient upon unfolding appear to be temperature dependent.
• 3.Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding.
• 4.Structural plasticity of proteins defines the position dependent effect of amino acid substitutions of the residues buried in the native state.
• 5.Small proteins have positive volume changes upon unfolding which suggests difference in balance between the cavity/void volume in the native state and the hydration volume changes upon unfolding as compared to the large proteins that have negative volume changes.

General significanceThe information provided here gives a better understanding and deeper insight into the role played by various factors in defining the volume changes upon protein unfolding. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Binding of glucagon to lipid bilayers

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 X 10(-6)-3.0 X 10(-8) M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 X 10(-7) M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 N epsilon-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 N epsilon-amino groups showed a transition in reactivity at 1.8 X 10(-7) M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 X 10(-7) M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10(-6) M, with free monomeric glucagon occurring at 3 X 10(-8) M. A trimerization constant of 4 X 10(13) M-2 at pH 7.5 and 37 degrees C was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry

Pressure perturbation calorimetry is a new technique that measures the heat change in a solution that results when the pressure above the solution is changed. When used in a differential calorimeter containing a dilute solution of solute in the sample cell and the corresponding buffer in the reference cell, the measured differential heat can be used to calculate the thermal coefficient of expansion of the partial volume of the solute, alpha. For proteins in dilute aqueous solution, alpha is dominated by a temperature-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to alpha can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, ribonuclease A, and T4 lysozyme) were examined carefully by this technique, allowing estimates of various volumetric parameters including the volume change resulting from thermal unfolding of each protein. For ribonuclease A, results obtained in both water and deuterium oxide led to an estimate of the accessible surface area of the native protein of approximately 45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to ribonuclease A led to changes in alpha, suggesting a burial of some surface area in the ligand-protein complex.     

Insertion of externally administered amyloid beta peptide 25-35 and perturbation of lipid bilayers

To understand the molecular basis and to prevent diseases such as Alzheimer's disease (AD), the targets of the triggering agent have to be elucidated. beta-Amyloid peptide (Abeta) is the major component of extracellular senile plaques characteristic of AD. For a very long time, the aggregated form of the Abeta was supposed to be responsible for the neurodegeneration that occurs in AD. Recently, the attention has been diverted to the monomeric or oligomeric forms of Abeta and their interaction with cellular targets. In our investigation, the physiological and medically important insertion of externally applied Abeta monomers into the bilayer of lipid vesicles is demonstrated. Abeta(25-35) has been localized in the region of the lipid alkyl chain, and it has a severe disordering effect on the lamellar order of the lipid bilayer. Both of these results are of biomedical relevance.     

Computational investigation of pressure profiles in lipid bilayers with embedded proteins

The distribution of surface tension within a lipid bilayer, also referred to as the lateral pressure profile, has been the subject of theoretical scrutiny recently due to its potential to radically alter the function of biomedically important membrane proteins. Experimental measurements of the pressure profile are still hard to come by, leaving first-principles all-atom calculations of the profile as an important investigative tool. We describe and validate an efficient implementation of pressure profile calculations in the molecular dynamics package NAMD, capable of distinguishing between internal, bonded and nonbonded contributions as well as those of selected atom groups. The new implementation can also be used in conjunction with Ewald summation for long-range electrostatics, improving the accuracy and reproducibility of the calculated profiles. We then describe results of the calculation of a pressure profile for a simple protein–lipid system consisting of melittin embedded in a DMPC bilayer. While the lateral pressure in the protein–lipid system is nearly the same as that of the bilayer alone, partitioning of the lateral pressure by atom type revealed substantial perturbation of the pressure profile and surface tension in an asymmetric manner.     

Thermotropic phase behaviour of lipid bilayers containing carotenoid pigment canthaxanthin: a differential scanning calorimetry study

In this study we address the problem of the effect of canthaxanthin on the thermotropic properties of lipid membranes formed with lipids which differ in the thickness of their hydrophobic core, size of polar heads or presence of the ester carbonyl group. For all the lipids a decrease in main transition enthalpy has been observed, indicating that canthaxanthin alters the membrane properties in its gel phase. The strongest influence of canthaxanthin on main phase transition and pretransition has been observed for the lipid having the thinnest hydrophobic region. Component analysis indicates a distinct cooperativity change, which most probably colligates with the formation of new thermotropic phases. The effect of canthaxanthin has been almost negligible in the case of phosphatidylethanolamines. The absence of the ester carbonyl group results in different thermotropic behavior, especially for low canthaxanthin concentrations. The effect of canthaxanthin is explained in terms of its organization within the membrane.     

Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22 degrees C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, delta A, at constant surface pressure, pi. At a given surface pressure, delta A increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, delta A, was strongly dependent on the surface pressure. The area increase, delta A, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, delta A/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The delta A/A vs. pi isotherms intersected at pi = 39.5 +/- 1 mN/m with delta A = O, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, chi b, in the monolayer (chi b greater than 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to charge repulsion effects, which at still higher molar ratios (chi b greater than 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of lipid bilayers

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Asymmetric lipid bilayers Response to multivalent ions

Asymmetric lipid bilayers are formed by adjoining the hydrocarbon chains of two different lipid monolayers at the air-water interface through an aperture in a teflon partition separating two aqueous phases. It is shown that the addition of Ca²⁺ or polysine to the compartment limited by a monolayer of the neutral lipids glycerol dioleate or phosphatidylcholine results in no modification of the resistance and stability of the membrane, whereas a drastic decrease in both parameters is elicited by the presence of these ions on the opposite compartment containing a monolayer of the negatively charged cardiolipin or phosphatidylserine. The surface-charge dependence of this phenomenon indicates the persistence of the asymmetric lipid distribution in the bilayer after its formation from two different monolayers.     

Condensed complexes and the calorimetry of cholesterol-phospholipid bilayers

A recent thermodynamic model describes a reversible reaction between cholesterol (C) and phospholipid (P) to form a condensed complex C(nq)P(np). Here q and p are relatively prime integers used to define the stoichiometric composition, and n is a measure of cooperativity. The present study applies this model to the scanning calorimetry of binary mixtures of cholesterol and saturated phosphatidylcholines, especially work by McElhaney and collaborators. These mixtures generally show two heat capacity peaks, a sharp peak and a broad peak. The sharp heat absorption is largely due to the chain melting transition of pure phospholipid. In the present work the broad heat absorption is attributed to the thermal dissociation of complexes. The best fits of the model to the data require the complex formation to be highly cooperative, with cooperativity n = 12. Detailed comparisons are made between model calculations and calorimetric data. A number of unusual features of the data arise naturally in the model. The principal discrepancy between the calculations and experimental results is a spurious calculated heat absorption peak. This discrepancy is related to the reported relative magnitudes of the integrated broad and sharp heat absorption curves.     

The role of the anticancer drug vinorelbine in lipid bilayers using differential scanning calorimetry and molecular modeling

Differential scanning calorimetry (DSC) has been employed to investigate the thermal changes caused by the anticancer alkaloid drug vinorelbine in dipalmytoylphosphatidylcholine (DPPC) bilayers. The total enthalpy change was increased by the presence of the drug molecule, indicating a partial interdigitation of the lipid alkyl chains. The presence of cholesterol in DPPC bilayers including vinorelbine induced an obstruction of the interdigitation, since cholesterol interrupts the upraise of enthalpy change. Vinorelbine's interdigitation ability and stabilizing properties with the active site of the receptor have been compared with those of similar in structure amphipathic and bulky alkaloid vinblastine. The obtained results may in part explain their similar mechanism of action but different bioactivity.     

High pressure infrared spectroscopy of lipid bilayers: new tests for interdigitation

High-pressure infrared spectroscopy is used to compare the barotropic behaviour of various interdigitated lipid bilayer systems (1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine, 1,3-dipalmitoyl-sn-glycero-2-phosphocholine and 1-palmitoyl-sn-glycero-3-phosphocholine) with non-interdigitated bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In the pressure range between 0 and 20 kbar, we have monitored the pressure dependence of the relative peak height intensity ratio of the chain methylene scissoring band, delta CH2, and its correlation field component band, delta'CH2. We demonstrate that this parameter, in conjunction with a visual inspection of the pressure-induced correlation field splittings of the methylene scissoring and rocking mode bands, can provide reliable indications of chain interdigitation.     

The influence of 1-alkanols and external pressure on the lateral pressure profiles of lipid bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.