Condensed complexes of cholesterol and phospholipids.

Mixtures of dihydrocholesterol and phospholipids form immiscible liquids in monolayer membranes at the air-water interface under specified conditions of temperature and 2-dimensional pressure. In recent work it has been discovered that a number of these mixtures exhibit two upper miscibility critical points. Pairs of upper critical points can be accounted for by a theoretical model that implies the cooperative formation of molecular complexes of dihydrocholesterol and phospholipid molecules. These complexes are calculated to be present in the membranes both above and below the critical points. Below the critical points the complexes form a separate phase, whereas above the critical points the complexes are completely miscible with the other lipid components. The cooperativity of complex formation prompts the use of the terminology condensed complex.     

Electronic structure calculation study of metal complexes with a phytosiderophore mugineic acid

Structural and thermodynamic properties of biologically important metal-mugineic acid complexes have been studied from the theoretical side in order to understand the metal-chelating mechanism of phytosiderophore mugineic acid at an atomic level. Density-functional theory methods combined with the polarizable continuum model (PCM) have been employed to obtain free energies of complex formation and redox potentials for metal-mugineic acid complexes in solution. It has been found that the free energies of complex formation calculated at the B3LYP/PCM level of theory are in moderate agreement with available experimental results. The inclusion of explicit water molecules interacting with the carboxylic groups in deprotonated mugineic acid through strong hydrogen-bonds is found to further improve the calculated free energies of complex formation.     

Interaction of N-myristoylethanolamine with cholesterol investigated in a Langmuir film at the air-water interface

The dramatic increase in the content of N-acylethanolamines (NAEs) having different acyl chains in various tissues when subjected to stress has resulted in significant interest in investigations on these molecules. Previous studies suggested that N-myristoylethanolamine (NMEA) and cholesterol interact to form a 1:1 (mol/mol) complex. In studies reported here, pressure-area isotherms of Langmuir films at the air-water interface have shown that at low fractions of cholesterol, the average area per molecule is lower than that predicted for ideal mixing, whereas at high cholesterol content the observed molecular area is higher, with a cross-over point at the equimolar composition. A plausible model that can explain these observations is the following: addition of small amounts of cholesterol to NMEA results in a reorientation of the NMEA molecules from the tilted disposition in the crystalline state to the vertical and stabilization of the intermolecular interactions, leading to the formation of a compact monolayer film, whereas at the other end of the composition diagram, addition of small amounts of NMEA to cholesterol leads to a tilting of the cholesterol molecules resulting in an increase in the average area per molecule. In Brewster angle microscopy experiments, a stable and bright homogeneous condensed phase was observed at a relatively low applied pressure of 2 mN.m(-1) for the NMEA:Chol. (1:1, mol/mol) mixture, whereas all other samples required significantly higher pressures (>10 mN.m(-1)) to form a homogeneous condensed phase. These observations are consistent with the formation of a 1:1 stoichiometric complex between NMEA and cholesterol and suggest that increase in the content of NAEs under stress may modulate the composition and dynamics of lipid rafts in biological membranes, resulting in alterations in signaling events involving them, which may be relevant to the putative cytoprotective and stress-combating ability of NAEs.     

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.     

Chemical activity of cholesterol in membranes

Measurements are reported for the rate constants for the release of cholesterol (and dihydrocholesterol) to beta-cyclodextrin from mixtures with phospholipids in homogeneous monolayers at constant pressure at the air-water interface. In each mixture, it is found that the release rate shows a sharp decrease as the cholesterol concentration in the monolayer decreases through a composition corresponding to the stoichiometry of a cholesterol-phospholipid complex. The stoichiometry of the complex was established previously by the position of a sharp cusp in the thermodynamic phase diagram of each mixture and also by a minimum in average molecular area versus composition measurements. A theoretical model used earlier to account for the phase diagrams predicts the chemical potential and chemical activity of cholesterol in these mixtures. The calculated chemical activity also shows a sharp change at the complex stoichiometry in homogeneous monolayers. The similarities in change of observed release rate and calculated chemical activity are expected from reaction rate theory where the release rate is proportional to the cholesterol chemical activity. The chemical activity of cholesterol as determined by complex formation between some phospholipids and cholesterol in the plasma membrane of cells may serve a regulatory function with respect to intracellular cholesterol transport and biosynthesis.     

Thermodynamic analysis of interaction of mitoxantrone with deoxytetranucleotide 5'-d(TpGpCpA) in the water solution based on 1H-NMR spectrophotometry

The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5'-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) 1H NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters delta H and delta S of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)2 play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.     

A model for the formation and interconversion of protein A-immunoglobulin G soluble complexes

We present a model for the formation and interconversion of the soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies. The basic elements of the model are developed from reported hydrodynamic and electron microscopic studies of these complexes (see accompanying companion paper), together with established structural and binding properties of IgG and SpA. The model includes specific symmetry and binding requirements for IgG-SpA combination, and a steric constraint between neighboring IgG molecules. We discuss how such a constraint could influence the assembly and distribution of equilibrium complexes. After formulating a convenient symbolism for representing IgG-SpA complexes, the suggested model is used to construct plausible structures for the four predominant complexes observed in moderate SpA excess. Distributions of these stable complexes at different IgG:SpA ratios, together with LeChatelier's principle and a straightforward thermodynamic derivation, are used to predict likely arrangements of equilibrium structures. Also, a scale model of the unique IgG4-SpA2 complex formed in IgG excess is constructed from reported x-ray diffraction and amino acid sequence data. An intuitive thermodynamic argument is used to show that the suggested steric constraint could cause the rather unprecedented reversible transformation of the four 7 to 15S complexes into the unique 17S complex. A computer simulation is used to predict equilibrium concentrations of the various proposed complexes at different IgG:SpA ratios. In support of the suggested structures, the calculated thermodynamic distributions agree surprisingly well with those measured with the ultracentrifuge. We point out how the proposed arrangements of the complexes, and in particular the 17S complex, can account for many of their novel properties, such as antigen-induced conformational changes. Reported differences in complement activation and precipitate formation by SpA complexes formed with antibodies from various species are also discussed with regard to possible differences in structural arrangements of the complexes.     

Influence of nonionic surfactant on aggregation state of scleroglucan in aqueous solution

The interactions between nonyl phenol polyethylene oxide and scleroglucan are investigated by turbidimetry, viscosimetry filterability tests and measurements of elastic modulus and adsorption. The phase diagrams of this ternary system have been established as a function of temperature and composition. It is shown that the surfactant molecules are adsorbed by the polymer at a low surfactant concentration, c_s; the adsorption induces a breaking down of the polymer aggregates and the filterability properties of the solutions are greatly improved. An excess of surfactant phase separation is observed by heating at a temperature that is a decreasing function of c_s. This is explained by the formation of a complex polymer-surfactant which has the same thermodynamic properties (lower critical solution temperature) as polyethylene oxide and the derived nonionic surfactants.     

Condensed complexes of cholesterol and phospholipids

There is overwhelming evidence that lipid bilayer regions of animal cell membranes are in a liquid state. Quantitative models of these bilayer regions must then be models of liquids. These liquids are highly non-ideal. For example, it has been known for more than 75 years that mixtures of cholesterol and certain phospholipids undergo an area contraction or condensation in lipid monolayers at the air-water interface. In the past 3 years, a thermodynamic model of "condensed complexes" has been proposed to account for this non-ideal behavior. Here we give an overview of the model, its relation to other models, and to modern views of the properties of animal cell membranes.     

The binary phase diagram of lecithin and cholesteryl linolenate

The condensed binary phase diagram of cholesteryl linolenate-egg yolk lecithin has been determined by polarizing light microscopy, differential scanning calorimetry and X-ray diffraction. On increasing the temperature lecithin forms rectangular, cubic and hexagonal liquid-crystalline structures into which varying amounts of cholesteryl linolenate are incorporated. As more cholesteryl linolenate is incorporated, the transition temperatures between different phases are lowered. The rectangular and cubic structures incorporate only small amounts of cholesteryl linolenate; the molar ratios, lecithin to cholesteryl linolenate, being 11:1 and 16:1, respectively. However, the hexagonal phase, in which the phosphorylcholine groups of the lecithin molecules form the core of the rod-like assembly of molecules, incorporates up to approximately 25% cholesteryl linolenate by weight, corresponding to a molar ratio 3:1. At higher concentrations, cholesteryl linolenate forms an excess phase and may be present as crystals, smectic or cholesteric liquid crystals, or as liquid oil, depending on the temperature. At higher temperatures, a large zone of a single isotropic liquid phase exists in which large amounts of lecithin are solubilized by the cholesterol ester. Up to 40% cholesteryl linolenate by weight, the transition temperatures between different phases are influenced by approximately 1% water (by weight) associated with egg lecithin.It is probable that the incorporated apolar cholesterol ester molecules are associated primarily with the apolar hydrocarbon chain region of the different lecithin structures. The resultant decrease in the observed transition temperatures would suggest an overall chain-disordering role for the incorporated cholesteryl linolenate molecules. The influence of cholesteryl linolenate on the thermodynamic stability of the different lecithin structures, together with the models suggested for the molecular orientations of cholesterol esters in the different liquid crystalline structures, may be relevant to the role of these lipids in more complex biological systems, particularly serum lipoproteins.     

Interaction of trypsin with multilamellar vesicles of soybean lipids

The pH dependence of complex formation of trypsin with multilamellar vesicles (MLV) of soybean lipids has been investigated. The lipids were characterized by the same phospholipid composition, but the content of other lipids differed. Decrease of pH or introduction of negatively charged components into the lipid samples increased trypsin content in the protein-lipid complexes. This suggests electrostatic interaction between the protein and soybean lipids. The dependence of trypsin activity in the complexes with MLV on their concentration and on the presence of an ionic detergent was studied. Trypsin-MLV interaction did not result in complete inactivation of the protein molecules. Moreover, the effects of dilution and addition of ionic detergent on trypsin activity were additive. Using a fluorescence technique, complex formation with MLV was found to stabilize trypsin molecules, preventing their autolysis.     

Recombinant models of lipoproteins. Apolipoprotein A-I/phosphatidylcholine/cholesterol complexes formed in a 2-chloroethanol-water mixture

Apolipoprotein A-I can spontaneously associate with phosphatidylcholine and cholesterol in 2-chloroethanol-water mixture. It was demonstrated, using a spin label technique, that dissolved molecules participate in complex formation. The apolipoprotein A-I/phosphatidylcholine/cholesterol complexes were isolated by gel chromatography. Complexes of three types were prepared and characterized: type A, large heterogeneous aggregates with molecular weight 600 000, sedimentation coefficient 10 S and the following molar composition - protein/phosphatidylcholine/cholesterol, 1:(70-100):(10-12); types B and C, with weight average molecular weights 140 000 and 110 000, average sedimentation coefficients 3.6 S and 1.7 S, respectively. Both types have the same molar composition - protein/phosphatidylcholine/cholesterol, 1:25:8. The dissimilar sedimentation coefficients between complexes B and C may be explained by the difference in the monomer/tetramer ratio (monomer molecular weight 50 000). The spin label sn-1-O-stearoyl-2-O-9'-spiro(4',4'-dimethyloxazolidine-3'-oxyl) heptadecanoylglycero-3-phosphocholine introduced into the complexes A and B showed different thermal properties of these complexes, which may be due to differences in the lipid-protein interactions.     

Conformational Analysis of Amphotericin B — Cholesterol Channel Complex

A molecular model of ionic channel formed by flexible molecules of amphotericin B and cholesterol is proposed. Complexes with axial symmetry from 5 to 11 were simulated. In contrast to the model of the channel formed from rigid molecules, flexible molecules form a tightly packed structure consolidated by both dispersive forces and intermolecular hydrogen bonds. Contributions of a lactone ring, polar heads, cholesterol and lipid environments to the global energy of the complex formation are discussed. Among the complexes capable of ionic transport, that of axial symmetry eight is preferable. Two types of complexes, differing by the number of intramolecular hydrogen bonds, are shown to be possible. Received: 25 April 1997/Revised: 20 November 1997     

Interactions of the lipopeptide antifungal iturin A with lipids in mixed monolayers

The miscibility and the interactions of the antifungal lipopeptide iturin A with lipids, DMPC and cholesterol, are studied in monolayers at the air/water interface and a comparison of the respective behaviour of iturin A and the biologically inactive methylated derivative MeTyr-iturin A is made. Each lipopeptide is miscible with anyone of the lipids. This behaviour is revealed by the dependence of the transition pressure upon composition and by deviations from the additivity rule of the mean molecular area. The thermodynamic properties of the mixed systems are studied by the method of Goodrich. The mixed monolayers are always more stable than the two separate components, subsequently there are interactions between the components. However, the excess free energy of mixing delta Gexm is positive for the iturin A/DMPC system which is an indication that the interactions between lipopeptide and lipid molecules are weaker than the interactions between the pure components themselves. This is compatible with the presence of self-associated lipopeptide molecules. However, delta Gexm is highly negative for the iturin A/cholesterol system giving evidence of the formation of a specific complex between iturin A and cholesterol which is not the case with the methylated derivative. These data are analysed in connection with previous results concerning the pore-forming properties of these lipopeptides and the lack of biological activity of MeTyr-iturin A.     

Characterization of two novel human small heat shock proteins: protein kinase-related HspB8 and testis-specific HspB9

Some binary mixtures of cholesterol and phospholipids in monolayers have thermodynamic phase diagrams with two upper miscibility critical points. This feature has been interpreted in terms of 'condensed complexes' between the phospholipid and cholesterol. The present work gives evidence for the formation of complexes with a common simple integral stoichiometry in binary mixtures of cholesterol and a series of five sphingomyelins where the amide-linked acyl chain length is varied. This indicates that these complexes have a distinct geometry even though they form a liquid phase.     

Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation

The reversibility of the binding of human apolipoprotein A-I (apo A-I) to phospholipid has been monitored through the influence of guanidine hydrochloride (Gdn-HCl) on the isothermal denaturation and renaturation of apo A-1/dimyristoylphosphatidylcholine (DMPC) complexes at 24 degree C. Denaturation was studied by incubating discoidal 1:100 and vesicular 1:500 mol/mol apo A-I/DMPC complexes with up to 7 M Gdn-HCl for up to 72 h. Unfolding of apo A-I molecules was observed from circular dichroism spectra while the distribution of protein between free and lipid-associated states was monitored by density gradient ultracentrifugation. The ability of apo A-I to combine with DMPC in the presence of Gdn-HCl at 24 degrees C was also investigated by similar procedures. In both the denaturation and renaturation of 1:100 and 1:500 complexes, the final values of the molar ellipticity and the ratio of free to bound apo A-I at various concentrations of Gdn-HCl are dependent on the initial state of the lipid and protein; apo A-I is more resistant to denaturation when Gdn-HCl is added to existing complexes than to a mixture of apo A-I and DMPC. There is an intermediate state in the denaturation pathway of apo A-I/DMPC complexes which is not present in the renaturation; the intermediate comprises partially unfold apo A-I molecules still associated with the complex by some of their apolar residues. Complete unfolding of the alpha helix and subsequent desorption of the apo A-I molecules from the lipid/water interface involve cooperative exposure of these apolar residues to the aqueous phase. The energy barrier associated with this desorption step makes the binding of apo A-I to DMPC a thermodynamically irreversible process. Consequently, binding constants of apo A-I and PC cannot be calculated simply from equilibrium thermodynamic treatments of the partitioning of protein between free and bound states. Apo A-I molecules do not exchange freely between the lipid-free and lipid-bound states, and extra work is required to drive protein molecules off the surface. The required increased in surface pressure can be achieved by a net mass transfer of protein to the surface; in vivo, increases in the surface pressure of lipoproteins by lipolysis can cause protein desorption.     

Thermodynamic description of oligonucleotide self-association in DNA concatamer structures

A scheme of equilibrium formation of concatamers by two different oligonucleotides has been considered. It is shown that in the general case, the dependence of the concentration of oligonucleotide components on temperature cannot be found in analytical form. Therefore, it is impossible to find the thermodynamic parameters of concatamer formation (ΔH ⁰, ΔS ⁰) and melting temperatures by analyzing the profiles of thermal denaturation of oligonucleotide complexes. An algorithm for numerical solution of implicit dependences has been developed. A number of approaches are considered that simplify the analysis of heat denaturation curves for concatamer complexes. It is shown that the dependence of the efficiency of concatamerization on temperature can be described analytically when duplex fragments have close stability and there is no cooperativity at the oligonucleotide junction. In this case, the dependence of melting temperature on thermodynamic parameters and oligonucleotide concentration has the same form as in the case of the duplex structure formed by a pair of non-self-complementary oligonucleotides. The ability of various model approaches to describe the experimental curves of concatamer heat denaturation is evaluated. For concatamer structures used as signal amplifiers in DNA hybridization analysis, a function is introduced that shows the relative contribution of a concatamer of given length to the magnitude of signal amplification. The dependence of the maximum of this function on the concentration of oligonucleotides, the thermodynamic characteristics of their complexes, and temperature has been determined. It is shown by the gel retardation assay that the function of the length distribution of concatamers qualitatively correlates with the experimental dependences.     

"Bridge" structures between nucleic acid molecules fixed in the structure of a liquid crystal

The binding of daunomycin and copper ions to poly(I).poly(C) molecules fixed in a particle of a liquid-crystalline dispersion was studied. A thermodynamic model of adsorption was developed, which makes it possible to describe the formation of complexes of a particular kind, "bridges" that connect adjacent nucleic acid molecules fixed in a liquid crystal. The bridges represent chelate complexes, which incorporate the molecules of the antibiotic daunomycin and copper ions. Equations describing the dependence of the concentration of these bridges in solution on the concentration of their constituents were derived. The family of dependences of experimental amplitudes of bands in CD spectra typical of "bridge" structures on the concentration of copper ions represents a set of S-shaped curves, and, as the concentration of daunomycin in solution increases, the level of saturation of these curves increases. The analysis of experimental data with the use of this model suggests that the structures of this type compete with daunomycin molecules for the binding sites on poly(I).poly(C). By using this model, the energies of formation of bridge structures were calculated.     

Effect of protein side chain amide group on the hydrogen-bond equilibrium in nucleobases studied by infrared and 13C-NMR spectroscopy

Infrared spectra of 1:1 hydrogen-bonded complexes formed by derivatives of adenine and model molecules bearing the protein side chain amide group have been measured in chloroform solution. From the temperature dependence of hydrogen-bond formation, thermodynamic data on these complexes are determined. On the basis of these data, it is shown that the complexes consist of cyclic heterodimers, those that use the adenine N(1)H bond being favoured. Similarly infrared and 13C-NMR spectroscopy reveals that uracil-amide cyclic heterodimers formed through the uracil 4-carbonyl group are predominant. All of these results predict that Watson-Crick hydrogen bonds in adenine-uracil base-pairs may be opened to some extent, as proved in this work. The possible biological importance of these observations is also discussed.     

Complexes in ternary cholesterol-phospholipid mixtures

Recent work by Veatch and Keller has described micron-scale liquid-liquid immiscibility in giant unilamellar vesicles composed of ternary mixtures of cholesterol, dipalmitoylphosphatidylcholine (DPPC), and dioleoylphosphatidylcholine (DOPC). Significantly, they do not observe micron-scale immiscibility in any of the three corresponding binary mixtures under the same conditions. It is shown here that this unexpected result can be accounted for by the formation of a complex between cholesterol and DPPC. The complex is miscible with DPPC and cholesterol, and immiscible with DOPC. A simple, idealized thermodynamic treatment of this model leads to theoretical ternary phase diagrams that are similar to the experimental diagram reported by Veatch and Keller. The model also accounts for significant qualitative features of the deuterium NMR spectra of these mixtures in bilayers.