Cholestane Spin Label Research Articles

Oxidation of Cholesterol and Formation of Cholesterol Hydroperoxides Decreases the Cholesterol Concentration at which Formation of Cholesterol Bilayer Domains and Cholesterol Crystals is Initiated in Phospholipid Bilayers

Recently we demonstrated that formation of cholesterol bilayer domains (CBDs) precedes formation of cholesterol crystals (CHCs) in cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) membranes (Mainali et. al., J. Phys. Chem. B, 117(2013)8994-9003). In the presented studies, we show that the oxidation of Chol in Chol/DMPC membranes induces formation of CBDs and CHCs at significantly lower Chol concentrations than in membranes without oxidation. To preserve compositional homogeneity throughout the membrane suspension we prepared the multilamellar liposomes with a rapid solvent exchange method. The oxidation of Chol was induced using a photosensitizer, rose Bengal, and the extent of oxidation (formation of Chol hydroperoxides) was measured using iodometric assay. To avoid formation of phospholipid hydroperoxides, which would interfere with our assay, the saturated phospholipid (DMPC) was used to prepare liposomes. Saturation recovery electron paramagnetic resonance with spin-labeled cholesterol analogues (androstane and cholestane spin labels) was used to detect the formation of CBDs and differential scanning calorimetry was used to detect the formation of CHCs. We conclude that formation of Chol hydroperoxides induces the formation of CBDs at Chol concentrations where previously CBDs were not present in non-oxidized membranes as well as the collapse of the already formed CBDs to CHCs, presumably outside the lipid bilayer.

A New Phase Boundary in Phosphatidylcholine/Cholesterol Bilayers (In the Dimyristoylphosphatidylcholine/Cholesterol Bilayer): EPR and DSC Studies

A new phase boundary, at cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) mixing ratio of ∼1, was observed by saturation-recovery electron paramagnetic resonance (EPR) spin-labeling method in a multilamellar suspension of DMPC and Chol prepared using a rapid solvent exchange method. With spin-labeled cholesterol analogues (androstane spin label [ASL] and cholestane spin label [CSL]) it was shown that the upper limit of the cholesterol concentration in the liquid-ordered phase of the DMPC membrane is ∼50 mol%, above which the excess of cholesterol forms the pure cholesterol bilayer domain (CBD). Thus, the phase boundary at a Chol/DMPC mixing ratio of ∼1 separates the region with a single liquid-ordered phase from the region with a coexisting liquid-ordered phase and the CBD. Because the pure cholesterol cannot form the free bilayer in the buffer, the CBD has to be supported by the surrounding phospholipid bilayer saturated with cholesterol. The next phase boundary is defined by the cholesterol solubility threshold (CST) that indicates the amount of cholesterol which can saturate the DMPC bilayer and form the CBD. The excess of cholesterol above the CST forms monohydrate cholesterol crystals which precipitate outside of the lipid bilayer. It was shown by differential scanning calorimetry (DSC) that the CST, which separates the region with a coexisting liquid-ordered phase saturated with cholesterol and the CBD from the region in which cholesterol crystals are formed, is located at the Chol/DMPC mixing ratio of ∼2 (∼66 mol% cholesterol). The extended phase diagram for the DMPC/cholesterol membrane, covering the region where the membrane is saturated and oversaturated with cholesterol, is proposed.

Phase Boundaries in Phosphatidylcholine Membranes Saturated and Oversaturated with Cholesterol

Conventional and saturation-recovery EPR along with differential scanning calorimetry (DSC) were used to determine the cholesterol/phosphatidylcholine (Chol/PC) mixing ratio at which cholesterol bilayer domains (CBDs) and cholesterol crystals (CR) start to form in dimyristoyl-PC (DMPC) and 1-palmitoyl-2-oleoy-PC (POPC) membranes. The Chol/PC mixing ratio was changed from 0 to 3. Lipid multilamellar dispersions investigated in this work were prepared using the rapid solvent exchange method to preserve compositional homogeneity throughout the suspension. EPR spin-labeling methods were used with spin-labeled cholesterol analogues (cholestane spin label [CSL] and androstane spin label [ASL]) as well as with a spin-labeled phospholipid (1-palmitoyl-2-(5-doxylstearoyl)phosphatidylcholine (5-PC)) to locate the phase boundary for CBD formation. DSC was used to locate the phase boundary for the formation of CRs. Results showed that in both DMPC and POPC membranes the CBD is already formed at a Chol/PC mixing ratio of 1, while CRs are formed only when the Chol/PC mixing ratio exceeds the value of 2. Previous results and data presented here indicate that when the Chol/PC mixing ratio increases, the PC bilayer becomes saturated with cholesterol first. This phospholipid bilayer, saturated with cholesterol, possesses unique physical properties. Increase in cholesterol content beyond the Chol/PC mixing ratio of 1 causes formation of the CBD, and further increase, beyond the Chol/PC mixing ratio of 2, causes formation of CRs (presumably outside of the membrane). Thus, the phase boundary at a Chol/PC mixing ratio of 1 separates the region with a single liquid-ordered phase and the region with a coexisting liquid-ordered phase and CBD. The cholesterol solubility threshold at a Chol/PC mixing ratio of 2 separates the region with a coexisting liquid-ordered phase and CBD from the region in which CRs are formed.

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.

Abstract: S3-29 MULTIPLE MUTATIONS PERTURB A SMALL NUMBER OF PATHWAYS IN AN HDL MUTAGENESIS SCREEN

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, β-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21–55°C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35°C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter 〈P2〉, without affecting the diffusion coefficients.Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D⊥ of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225–233, and 982, 196–204).

Influence of High Orientational Order on the Shape of the Echo Response from a Hahn Pulse Sequence

The amplitude modulations in the simulations of the Hahn echo responses from cholestane spin labels in samples characterized by a high degree of orientational order are shown to arise from the use of “soft” pulses. Soft pulses have a limited spectral range and cover only a small portion of the CW-ESR spectra, so that not all the spins are on-resonance. The magnetization vectors of the off-resonance spins only partially tilted away from the laboratoryzaxis, the direction of the applied static magnetic field. They thus contribute oscillating components to the magnetization in thexyplane. The contribution from the off-resonance spins to the Hahn echo formation is significant in highly oriented samples, but cancels out in samples exhibiting a small degree of order. Experimental echo responses obtained from CSL molecules embedded in rigid matrices of eggPC bilayers and the liquid crystalline materials ZLI and MBBA confirm the theoretical predictions.

Effects of Fumonisin B1and (Hydrolyzed) Fumonisin Backbone AP1on Membranes: A Spin-Label Study

Electron spin resonance (ESR) spectroscopy and spin label techniques have been used to study the effects of fumonisin B1(FB1) and hydrolyzed fumonisin backbone (AP1) on the structural and dynamic properties of phosphatidylcholine membranes at the molecular level. Multilamellar liposomes consisting of dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EYPC) were used. Six different nitroxide spin labels were used to determine what effects FB1may impart on the ordering and mobility of lipids in membranes. The experimental results disclose the following: (1) In the fluid phase membrane, FB1significantly increases the fluidities ofn-doxylstearic acid (SA) spin labels (SL) attached to carbons 5 and 7, which disorders the alkyl chains and perturbs the surface region of the bilayer; by comparison, minimal effects were detected near the center of the bilayer. (2) In the gel phase, FB1and AP1imparts marked rigidifying effects on membrane fluidity, which enlarges the change in ordering on the phase transition even further. (3) FB1also restricts the mobility of the (rigid) cholestane spin label. (4) A reduction in mobility of the tempo-stearate spin label suggests that the tricarballylic acid (TCA) moieties of FB1might mimic the structure of polar headgroups in phospholipids. The present results may provide additional mechanisms to elucidate the toxicological activities of the fumonisins.

Molecular Motion of the Cholestane Spin Label in a Multibilayer in the Gel Phase Studied Using Echo-Detected EPR

Echo-detected EPR spectra of the cholestane spin label in a macroscopically oriented lipid multibilayer have been obtained at temperatures between 50 and 210 K. The drastic temperature dependence which the spectra reveal was attributed to relaxation induced by the orientational oscillations of the spin-label molecule. The scheme of computer simulations of echo-detected spectra for the model of uniaxial orientational oscillations (librations) is described in detail. The simulated spectra show a remarkable dependence on the orientation of the motional axis in the molecular frame. The simulation and experimental results agree when the projection of this axis in the plane of the nitroxide fragment makes an angle of 70° ± 10° with the direction of the NO bond. This was attributed to librational motion of the label about the long axis of the cholestane moiety.

A computer simulation study of the relation between lipid and probe behaviour in bilayer systems

Computer simulations are presented of the behaviour of elongated probe molecules anchored to the interface of bilayers of dipalmitoylphosphatidylcholine (DPPC) above the phase transition of the hydrocarbon chains. The simulations mimic the behaviour of the fluorescent probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and Cholestane spin label (CSL) in the DPPC bilayers. In contrast to any experimental technique the simulations follow the behaviour of both the lipid molecules and the probe within the bilayer structure. Thus the relation between the behaviour of the probe molecules and the order and dynamics of the lipid chains can be studied in detail. We find that the presence of probe molecules, at the low concentrations used experimentally, causes only a marginal perturbation in the intrinsic properties of the lipid chains. The simulations presented substantiate the conventional prescription for describing the orientational behaviour of probe molecules in lipid bilayers in terms of a local effective orienting potential. They show further that the potential arises from the confinement of the probe molecules between long segments of lipid chains in elongated free-volume cavities within the bilayer structure. Consequently the description of the rotational dynamics needs to be refined in order to take into account the combined effect of the restricted free rattling motions of the probes within the free-volume cavities and the orientations of the cavities themselves relative to the normal to the bilayer plane. The time scale of the motions of the cavities within the bilayer is determined by the rotational motions of long segments of the lipid chains. These observations justify the use of rigid probe molecules such as TMA-DPH and Cholestane spin labels for monitoring the orientational order and dynamics in lipid bilayer systems.

A computer simulation study of probe molecule behaviour in lipid bilayer systems

Computer simulations are presented of the behaviour of elongated probe molecules anchored to the interface of lipid bilayers above the phase transition of the hydrocarbon chains. The simulations thus mimic the behaviour of the fluorescent probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and cholestane spin label in lipid systems. In contrast to any experimental technique the simulations follow the behaviour of both the lipid molecules and the probe within the bilayer structure. Thus the relation between the behaviour of the probe molecules and the order and dynamics of the lipid chains can be studied in detail. We find that the presence of probe molecules at the low concentrations used experimentally causes only a marginal perturbation in the intrinsic properties of the lipid chains. The simulations presented support for the conventional prescription for describing the orientational behaviour of probe molecules in lipid bilayers in terms of a local effective orienting potential. Th...

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.

A computer simulation study of the relation between lipid and probe behaviour in bilayer systems

Computer simulations are presented of the behaviour of elongated probe molecules anchored to the interface of lipid bilayers above the phase transition of the hydrocarbon chains. The simulations thus mimic the behaviour of the fluorescent probe 1-(4-(trimethylammonio)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and Cholestane spin label in lipid systems. In contrast to any experimental technique the simulations follow the behaviour of both the lipid molecules and the probe within the bilayer structure. Thus, the relation between the behaviour of the probe molecules and the order and dynamics of the lipid chains can be studied in detail. We find that the presence of probe molecules, at the low concentrations used experimentally, causes only a marginal perturbation in the intrinsic properties of the lipid chains. The simulations presented support the conventional prescription for describing the orientational behaviour of probe molecules in lipid bilayers in terms of a local effective orienting potential. They indicate, however, that the potential arises from the confinement of the probe molecules between long segments of lipid chains in elongated free-volume cavities within the bilayer structure. In this sense the orienting potential concept needs to be refined in order to take into account the combined effect of the restricted free rattling motions of the probes within the free-volume cavities and the orientations of the cavities themselves relative to the normal to the bilayer plane. The time scale of the motions of the cavities within the bilayer is determined by the rotational motions of long segments of the lipid chains. These observations justify the use of rigid probe molecules such as TMA-DPH and Cholestane spin labels for monitoring the orientational order and dynamics in lipid bilayer systems.

Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behavior of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.

The macroscopic organization of reconstituted M13 coat protein-phospholipid systems. An EPR spectroscopy and polarizing microscope study

The coat protein of the bacteriophage M13 in the α-helical state is reconstituted in macroscopically oriented systems of dioleoylphosphatidylcholine that are prepared by squeezing the reconstituted material between glass plates. The coat protein dramatically influences the macroscopic orientation of the multibilayers, as is investigated by polarizing microscopy and EPR spectroscopy of the cholestane spin label embedded in the bilayers. It is found that with increasing amounts of protein the spontaneous macroscopic orientation of the reconstituted system decreases. This effect is proposed to be due to an increase of the apparent viscosity of the lipid-protein systems with increasing amounts of protein. This is assumed to arise from a sticky effect of the C- and N-terminal protein parts that extend into the aqueous phase between the bilayers.

Translational diffusion in a smectic-A phase by electron spin resonance imaging: The free-volume model

The method of dynamic imaging of diffusion (DID)-ESR (electron spin resonance) has been utilized to study the anisotropy of translational diffusion of spin probes in the smectic A phase of a eutectic liquid crystal, S2. In particular, the nearly spherical perdeuterated-TEMPONE (PDT) and the rigid and elongated cholestane spin label (CSL) molecules were studied. Whereas D⊥ (the coefficient of diffusion perpendicular to the nematic director) showed simple Arrhenius dependence for both probes, diffusion parallel to the director displayed two different temperature regimes with a changeover of D∥ at t*≊26–27 °C. The regime above (below) t* is characterized by weak (strong) translational ordering. For CSL the ratio D⊥/D∥<1 above t* which indicates nematiclike behavior, but below t* the behavior is more smecticlike, i.e., D⊥/D∥≳1; for PDT D⊥/D∥≳1 over the whole temperature range. A free volume model is developed to interpret the activation energies associated with D⊥ and D∥ (i.e., E⊥ and E∥) in terms of the orientational and translational order parameters for the smectic phase and those for the spin probes. Also included are the variation of the compressibility across the smectic layer and the length of the probe relative to that of the thickness of the smectic layer. The fact that above t* E∥/E⊥ is unity for CSL but a little greater that unity for PDT is interpreted as due to the weaker coupling of the larger CSL molecule to the weak translational ordering and compressibility variation. Below t*, E∥/E⊥ becomes 1.52 and 1.80 for CSL and PDT, respectively, which may be interpreted in terms of enhancement of these smectic features. The free volume model may be used to analyze E∥ and E⊥ for self-diffusion and for a wide range of spin probes, including such very small probes like methane, as a function of the key parameters.

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847–851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.

The effects of phosphatidylethanolamine and phosphatidylglycerol headgroups on bilayer dynamics: a slow-motion ESR study

A study of the effect of phosphatidylethanolamine and phosphatidylglycerol headgroups on the behaviour of cholestane spin-label (CSL) molecules in three lipid bilayer configurations: planar multibilayers, multilamellar liposomes and small unilamellar vesicles (SUV) is presented. The results are compared with those obtained from the corresponding configurations of phosphatidylcholines with identical acyl chain composition. A comparison of the parameters describing the orientational order and dynamics of CSL in palmitoyloleoylphosphatidyl-ethanolamine (POPE) and palmitoyloleoylphosphatidylcholine (POPC) bilayer configurations indicates that the hydration of the lipid headgroups is the most important factor determining the rates of rotational motion. The orientational order of CSL is found to be lower in fully hydrated multilamellar liposomes and SUVs of negatively charged dioleoylphosphatidylglycerol (DOPG), than in the same configurations of the zwitterionic dioleoylphosphatidylcholine (DOPC). However, the rotational diffusion is not affected by the difference in the lipid headgroup. Apparently, the electrostatic repulsion in DOPG multilamellar liposomes and SUVs causes the orientational order to decrease, but without affecting the rates of rotational diffusion.

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, β-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21–55°C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35°C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter 〈 P 2 〉, without affecting the diffusion coefficients.Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D ⊥ of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225–233, and 982, 196–204).

Interaction between glycophorin and a spin-labeled cholesterol analogue in reconstituted dimyristoylphosphatidylcholine bilayer vesicles

The interaction between glycophorin and a spin-labeled cholesterol analogue has been investigated by EPR spectroscopy. In vesicles which were reconstituted by the freeze and thaw technique, direct evidence was obtained for a reorganisation of the membrane at low protein content (protein/lipid ratio less than 1:300). From the spin exchange interaction we were able to show a protein-induced clustering of the steroid in fluid and in gel state membranes. Tryptic cleavage of the complete N-terminus of glycophorin vanishes the effect. Whereas the removal of the sialic acid residues by neuraminidase digest had no influence on the EPR spectra. The interaction seems to be cholestane spin label specific since it was not observed with an androstane spin-label.

A planar rotor model for cholestane spin label motion in phospholipid multibilayers with high order

An EPR study of a macroscopically oriented multibilayer system of DMPC/cholesterol (2:1), order parameter S≈1, is presented. The conventional cholestane spin label 14N-CSL as well as its isotopically substituted analogue 15N-CSL were used in the experiments. The temperature range studied, −18 to 18° C, covered the rigid-limit and the slow-motion regime. The experimental spectra were simulated by a numerical solution of the stochastic Liouville equation using Padé approximant techniques. As the ordering of the CSL molecules in this bilayer system is virtually indistinguishable from perfect alignment of the long molecular axes along the director (the normal to the multibilayer plane) the planar rotor model was used to describe the reorientational motions. Thus the molecules were taken to possess only a single rotational degree of freedom about their long axes. The analysis of the slow-motion spectra reveals that the CSL molecules undergo a random rotational jump motion rather than small-step Brownian diffusion. The use of both the 14N-CSL and 15N-CSL greatly improves the reliability of the spectral simulations. Furthermore the use of the 15N-CSL molecules affords a unique mapping of the orientational distribution of the molecules in the rigid-limit spectra in this system, in contrast to the information obtained with the 14N-CSL spectra.