Effect of the phase state of lipids on their interaction with cobra cytotoxin

Interaction between cytotoxin of the Central Asia cobra venom and dimiristoylphosphatidylcholine bilayer depending on its phase state was studied by ESR with spin label. A conclusion can be drawn that the efficiency of cytotoxin effect on the membranes depends on their phase state. Cytotoxin molecules are incorporated into myophile region of the bilayer, only if the latter is in the liquid crystal state. The interaction between cytotoxins and lipids of the bilayer in a gel state is in the main conditioned by electrostatic forces.     

Molecular dynamics simulation of a phospholipid membrane

We present the results of molecular dynamics (MD) simulations of a phospholipid membrane in water, including full atomic detail. The goal of the simulations was twofold: first we wanted to set up a simulation system which is able to reproduce experimental results and can serve as a model membrane in future simulations. This goal being reached it is then further possible to gain insight in to those properties that are experimentally more difficult to access. The system studied is dipalmitoylphosphatidylcholine/water, consisting of 5408 atoms. Using original force field parameters the membrane turned out to approach a gel-like state. With slight changes of the parameters, the system adopted a liquid-crystalline state. Separate 80 ps runs were performed on both the gel and liquid-crystalline systems. Comparison of MD results with reliable experimental data (bilayer repeat distance, surface area per lipid, tail order parameters, atom distributions) showed that our simulations, especially the one in the liquid-crystalline phase, can serve as a realistic model for a phospholipid membrane. Further analysis of the trajectories revealed valuable information on various properties. In the liquid-crystalline phase, the interface turns out to be quite diffuse, with water molecules penetrating into the bilayer to the position of the carbonyl groups. The 10–90% width of the interface turns out to be 1.3 nm and the width of the hydrocarbon interior 3.0 nm. The headgroup dipoles are oriented at a small angle with respect to the bilayer plane. The resulting charge distribution is almost completely cancelled by the water molecules. The electron density distribution shows a large dip in the middle of the membrane. In this part the tails are more flexible. The mean life time between dihedral transitions is 20 ps. The average number of gauche angles per tail is 3.5. The occurrence of kinks is not a significant feature.Abbreviations MD molecular dynamics - DPPC dipalmitoylphosphatidylcholine - SPC simple point charges - DPPE dipalmitoylphosphatidylethanolamine Correspondence to: H. J. C. Berendsen     

Direct evidence of nitrogen coupling in the copper(II) complex of bovine serum albumin by S-band electron spin resonance technique

ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra.     

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

ESR center line shifts during phase transitions of spin-labeled dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl ethanolamine

The position H_o of the ESR central absorption maximum of a spin-labeled phospholipid dispersion was recorded in the manner of a g-factor; γ=hν/βH_o. The point H_o corresponds to the center line zero crossing point of the first derivative ESR spectrum. A 5-doxyl derivative of stearic acid was incorporated into dispersions of dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl ethanolamine formed either by shaking a lipid film in the presence of buffered saline or by dialysis of label and lipid in 2-chloroethanol against buffered saline. It was found that during the gel to liquid crystal phase transition, the decrease in the order parameter, S, ranged from about .045 to .06 and the increase in the parameter γ ranged from about .0001 to .0002 depending upon the lipid and preparation method. In all cases examined, the changes could be associated with the lipid phase transition. A simple model based on rapid anisotropic motion indicates that about a 38% decrease in line width accounts for the observed shift in γ.     

Models for coordination site of cytochrome P-450, characterization of hemin-thiolato complexes with S, O, and N donor ligands by electronic absorption and electron spin resonance spectra

Bisthiolato-hemin complexes exhibiting "two split Soret bands" at 370 and 460 nm, classified into "hyperporphyrin spectrum" was prepared with naturally occurring porphyrins (Fe(III)protoporphyrin IX and its dimethyl ester), thioglycolate esters, and tetramethylammonium hydroxide in organic solvents. The structure of the complexes was characterized by electronic absorption and electron spin resonance (ESR) spectrometries. These complexes were stable under air at room temperature, their apparent half-lives being about 30 min monitored by the intensities of the two Soret bands. Thus the bisthiolato-hemin complex containing thioglycolate ester was shown to be a model for the cytochrome P450(P450)-thiolato binding complex. Ligand exchange reactions of the bisthiolato-hemin complex with imidazole or methanol indicated that the intermediate species are stabilized as thiolato-hemin-imidazole or -methanol complexes. The latter intermediate complex was suggested to be a good model for low-spin ferric P450 as characterized by distinct beta- and alpha-bands at 530 and 560 nm, respectively, as well as a single Soret peak at approximately 410 nm. The result of the analysis on ESR g values and crystal field parameters for the bisthiolato-hemin, thiolato-hemin-imidazole, and thiolato-hemin-oxygen ligand complexes comparing with those for P450 itself and the ligand binding complexes revealed that the sixth ligand trans to the fifth thiolato ligand of the low-spin ferric P450 can be an oxygen atom of water molecule.     

Deuterium order parameters in relation to thermodynamic properties of a phospholiped bilayer. A statistical mechanical interpretation.

The physical properties of bilayers of dipalmitoyl-3-sn-phosphatidylcholine are analyzed in terms of a statistical model proposed by Marcelja (S. Marcelja (1974), Biochim. Biophys. Acta 367, 165). The model is used to calculate the segmental order parameters of the hydrocarbon chains, the transition temperature of the crystalline leads to liquid crystalline phase transition, the entropy change of the transition, the bilayer thickness, and the linear thermal expansion coefficient. The theoretical predictions are in excellent agreement with experimental results obtained by deuterium magnetic resonance, differential scanning calorimetry, and X-ray diffraction. The model yields the probabilities of trans and gauche conformations and also those of more specific conformational defects like kinks or jogs.     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.     

Structural Mechanics of DNA Wrapping in the Nucleosome

Experimental X-ray crystal structures and a database of calculated structural parameters of DNA octamers were used in combination to analyse the mechanics of DNA bending in the nucleosome core complex. The 1kx5 X-ray crystal structure of the nucleosome core complex was used to determine the relationship between local structure at the base-step level and the global superhelical conformation observed for nucleosome-bound DNA. The superhelix is characterised by a large curvature (597°) in one plane and very little curvature (10°) in the orthogonal plane. Analysis of the curvature at the level of 10-step segments shows that there is a uniform curvature of 30° per helical turn throughout most of the structure but that there are two sharper kinks of 50° at ± 2 helical turns from the central dyad base pair. The curvature is due almost entirely to the base-step parameter roll. There are large periodic variations in roll, which are in phase with the helical twist and account for 500° of the total curvature. Although variations in the other base-step parameters perturb the local path of the DNA, they make minimal contributions to the total curvature. This implies that DNA bending in the nucleosome is achieved using the roll-slide-twist degree of freedom previously identified as the major degree of freedom in naked DNA oligomers. The energetics of bending into a nucleosome-bound conformation were therefore analysed using a database of structural parameters that we have previously developed for naked DNA oligomers. The minimum energy roll, the roll flexibility force constant and the maximum and minimum accessible roll values were obtained for each base step in the relevant octanucleotide context to account for the effects of conformational coupling that vary with sequence context. The distribution of base-step roll values and corresponding strain energy required to bend DNA into the nucleosome-bound conformation defined by the 1kx5 structure were obtained by applying a constant bending moment. When a single bending moment was applied to the entire sequence, the local details of the calculated structure did not match the experiment. However, when local 10-step bending moments were applied separately, the calculated structure showed excellent agreement with experiment. This implies that the protein applies variable bending forces along the DNA to maintain the superhelical path required for nucleosome wrapping. In particular, the 50° kinks are constraints imposed by the protein rather than a feature of the 1kx5 DNA sequence. The kinks coincide with a relatively flexible region of the sequence, and this is probably a prerequisite for high-affinity nucleosome binding, but the bending strain energy is significantly higher at these points than for the rest of the sequence. In the most rigid regions of the sequence, a higher strain energy is also required to achieve the standard 30° curvature per helical turn. We conclude that matching of the DNA sequence to the local roll periodicity required to achieve bending, together with the increased flexibility required at the kinks, determines the sequence selectivity of DNA wrapping in the nucleosome.     

The effects of A23187 on the phospholipid phase transition of large unilamellar vesicles (LUVs) as detected by ultrasound spectroscopy

The effect of the hydrophobic Ca2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4: 1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG] membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, alpha lambda, was determined with a double crystal acoustic interferometer, as a function of temperature and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature (Tm) of the LUV suspensions remained near 42.0 degrees C, while the amplitude of alpha lambda at the phase transition increased dramatically. At Tm the relaxation frequency, where alpha lambda max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.     

Electron spin resonance and electron-spin-echo study of oriented multilayers of L alpha-dipalmitoylphosphatidylcholine water systems.

A detailed electron spin resonance (ESR) study of spin-labeled-oriented multilayers of L alpha-dipalmitoylphosphatidylcholine (DPPC) water systems for low water content (2-10% by weight) is reported with the purpose of characterizing the dynamical and structural properties of model membrane systems. Emphasis is placed on the value of combining such experiments with detailed simulations based on current slow-motional theories. Information is obtained regarding ordering and anisotropic rotational diffusion rates via ESR lineshape analysis over the entire motional range, from the fast motional region through the moderately slow and slow to the rigid limit. This includes the low-temperature gel phase, the liquid crystalline L alpha (1) phase and what appears to be a third high-temperature phase above the L alpha phase. Cholestane (CSL) and spin-labeled DPPC (5-PC, 8-PC, and 16-PC) have been used to probe different depths of the bilayer. While CSL and 5-PC both reflect the high ordering of the bilayer close to the lipid-water interface, CSL appears to be located close enough to the water for the nitroxide to be involved in hydrogen bonding with water molecules. 16-PC reflects the relatively low ordering near the tail of the hydrocarbon chain in the bilayer. Quantitative estimates of ordering and motion are obtained for these cases. The results from CSL indicate that close to the lipid-water interface the DPPC molecule is oriented approximately perpendicular to the bilayer in these low water-content systems. However, all three labeled lipid probes indicate that the hydrocarbon chain of DPPC may be bent away from the bilayer normal by as much as 30 degrees and this evidence is stronger at low temperatures. When cholesterol is added to the DPPC-water system at a concentration greater than or equal to 2.5 mol %, the ordering is greatly increased although the rotational diffusion rate remains almost unaffected in the gel phase. Electron spin echoes (ESE) are observed for the first time from oriented lipid-water multilayers. Results obtained from cw ESR lineshape analysis are correlated with data from ESE experiments, which give a more direct measurement of relaxation times. These results indicate that for detection of very slow motions (close to the rigid limit) ESE experiments are more sensitive to dynamics than continuous wave ESR for which inhomogeneous broadening becomes a major problem.     

Multiequilibrium binding of a spin-labeled local anesthetic in phosphatidylcholine bilayers

The equilibria among spin-labeled amine local anesthetic species in dioleoylphosphatidylcholine liposomes at an anesthetic: lipid mole ratio of 1:100 are investigated. Electron spin resonance (ESR) spectra demonstrate that anesthetic mobility within the bilayer is charge-dependent, with the uncharged species the more mobile. Partition coefficient measurements confirm ESR evidence that changes in anesthetic mobility represent anesthetic-phospholipid interaction and not changes in bilayer fluidity. Spin-exchange attenuation experiments show that anesthetics within the bilayer are accessible to the aqueous medium. Dependence of tertiary-amine anesthetic pK on dielectric constant has been used to estimate the interfacial pK. We propose a model of equilibria among species of the tertiary amine anesthetic in the aqueous medium and those intercalated in the bilayer, including a species electrostatically bound to the lipid phosphate. Using experimentally determined equilibrium constants, the model provides the binding constant between the electrostatically bound and unbound cationic anesthetics within the bilayer. The model stimulates the pH dependence of the mobile fraction of total anesthetic population determined by subtraction techniques on experimental ESR spectra.     

Using 31P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using 31P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic 31P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the 31P chemical shift anisotropy (CSA) and spin-spin relaxation times (T2), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.     

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.     

Orientational and motional properties of copper (II) complexes of dibenzotetraaza [14]annulenes in lipid bilayers: an ESR study.

ESR studies of two copper(II) complexes of substituted dibenzotetraaza [14]annulenes, CuL and CuLA, in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EYPC) are reported. Our data show that both complexes partition into the membranes and that the rotational motion of CuL is faster than CuLA. Analysis of the ESR spectra of these complexes in DMPC vesicles indicate that the Cu-motion parameter, which is a measure of the degree of resolution of the nitrogen hyperfine structure, changes abruptly at the main phase transition. At 1 mole %, both complexes lowered the fluid/gel phase transition temperature by 2 degrees C as measured by the Cu-motion parameter. A gradual change of the Cu-motion parameter is observed in EYPC liposomes over the same temperature range. ESR spectra of both CuL and CuLA in oriented membranes reveal that both complexes are well oriented with the plane of the complex perpendicular to the bilayer surface.     

Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.     

Homology modeling of human CCR5 and analysis of its binding properties through molecular docking and molecular dynamics simulation

In this study, homology modeling, molecular docking and molecular dynamics simulation were performed to explore structural features and binding mechanism of some inhibitors of chemokine receptor type 5 (CCR5), and to construct a model for designing new CCR5 inhibitors for preventing HIV attachment to the host cell. A homology modeling procedure was employed to construct a 3D model of CCR5. For this procedure, the X-ray crystal structure of bovine rhodopsin (1F88A) at 2.80? resolution was used as template. After inserting the constructed model into a hydrated lipid bilayer, a 20ns molecular dynamics (MD) simulation was performed on the whole system. After reaching the equilibrium, twenty-four CCR5 inhibitors were docked in the active site of the obtained model. The binding models of the investigated antagonists indicate the mechanism of binding of the studied compounds to the CCR5 obviously. Moreover, 3D pictures of inhibitor-protein complex provided precious data regarding the binding orientation of each antagonist into the active site of this protein. One additional 20 ns MD simulation was performed on the initial structure of the CCR5-ligand 21 complex, resulted from the previous docking calculations, embedded in a hydrated POPE bilayer to explore the effects of the presence of lipid bilayer in the vicinity of CCR5-ligand complex. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.     

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.     

Interaction of cytochrome P-450 with phospholipids in dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol mixtures

ESR and microcalorimetry methods were employed to investigate the thermotropic properties and structure of proteoliposomes that incorporate cytochrome P450 and DMPC-DMPG binary mixtures depending on cytochrome P450 content and phospholipid composition. The microcalorimetry data demonstrated that the incorporation of cytochrome P450 into the phospholipid mixture resulted in bilayer thermal stabilization. The maximum shift of the temperature and proteoliposome transition enthalpy were achieved at the protein/lipid molar ratio of 1:1000 in almost equimolar phospholipid mixture. Using fatty acids that were spin-labeled at different positions (C5, C12, C16), it has been shown that the incorporation of cytochrome P450 into lipid mixtures containing 0-100% DMPG decreases C12 and C16 mobility and increases the C5 order parameter at transition phase (30 degrees C) and liquid crystal phase (37 degrees C) of bilayer. The maximum alteration amplitude of the probes used was not characteristic for the separate DMPC and DMPG but rather for the mixture at the molar ratio close to equimolar value. It is proposed that cytochrome P450 incorporation into the binary mixture initiated the formation of the bilayer crystal-like phase.     

X-ray diffraction analysis of internal wool lipids

Polarised optical microscopy (POM) and X-ray diffraction techniques were applied to intercellular lipids extracted from wool to study their structural arrangement in order to determine their role in the diffusion properties of wool fibre. Intercellular wool lipids (IWL) arranged as concentrated liposomes were shown to be a good intercellular lipid model, allowing their study by X-ray diffraction techniques. The results confirm that intercellular lipids of wool fibre are organised in a lamellar structure of 5.0–8.0 nm width, termed β-layer, which had been assumed to be lipids arranged as a bilayer. Structurally, internal wool lipids are distributed at least in two domains at low temperatures: an ordered phase made up of ceramides and free fatty acids (FFA) alone, arranged in crystal orthorhombic states separately, and a liquid crystal state when mixed together. At 40 °C there is a reversible phase transition produced by the melt of the crystal orthorhombic states, whereas the liquid crystal state remains until 65 °C.