An ESR Spin label study of structural and dynamical properties of oriented lecithin-cholesterol multibilayers.

Oriented dipalmitoyllecithin-cholesterol multibilayers with 11% water have been studied with the cholestane spin label. From the ESR spectra the order parameters and the mobility of the spin label about its long axis have been calculated. The results on pure lecithin multibilayers indicate a transition from gel to liquid crystalline phase at 52 plus or minus 2 degrees C. In the gel phase the lecithin alkyl chains are highly ordered, but tilted with respect to the normal to the bilayers by about 25 degrees. Above 52 degrees C the tilt disappears and the mobility of the cholestane spin label increases, indicating an increase of mobility of the lecithin alkyl chains. When cholesterol is added, below about 52 degrees C a decrease of order is found. Furthermore, already small cholesterol contents (smaller than or equal to 10 mole %) remove the tilt. Above about 52 degrees C cholesterol improves the order by decreasing the amplitude of the librational motions. Cholesterol lowers the transition temperature of the system and reduces the mobility of the lecithin alkyl chains in the liquid crystalline phase. However an increase in mobility is found at cholesterol contents up to 10 mole %. A very broad phase transition is observed at 50 mole % cholesterol. In all systems an increase in temperature results in a reduction of order through an increase of the amplitude of the librational motions of the molecules. The librational motions are to some extent cooperative. The asymmetry of the order matrix is found to be a measure for the lateral ordering. Cholesterol increases the lateral ordering, indicating that the flat cholesterol molecules orient parallel to each other.     

Slow-motion ESR of cholestane spin labels in planar multibilayers of diacyldigalactosyldiglycerides

The orientation and restricted motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into planar multibilayers of diacyldigalactosyldiglycerides extracted from the thylakoid membranes of chloroplasts from different plant leaves has been studied. The experimental ESR spectra were simulated in terms of the slow-tumbling ESR formalism of Freed and co-workers (Polnaszek, C.F., Bruno, G.V. and Freed, J.H. (1973) J. Chem. Phys. 58, 3185–3199). The analysis shows that the degree of orientational order is low. The spin label molecules undergo a faster reorientational motion about their long molecular axes than perpendicular to them. At room temperature the reorientational rate around the long molecular axis falls within the fast-motional limit, while the reorientation rate of the long axis itself corresponds to the slow-tumbling regime. The results indicate that the motion of the labels in bilayers of diacyldigalactosyldiglycerides is considerably slower than that of the same label incorporated into bilayers of saturated phosphatidylcholines above the main phase transition. Differences between bilayers of diacyldigalactosyldiglycerides extracted from different plant membranes have been observed.     

Thermal effects on motion and orientation of the cholestane spin label in planar multibilayers of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine

A detailed picture of the orientation and restricted motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) in planar multibilayers of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine has been recorded by simultaneous simulation of ESR spectra obtained with the magnetic field parallel and perpendicular to the bilayers (Shimoyama, Y., Eriksson, L.E.G. and Ehrenberg, A. (1978) Biochim. Biophys. Acta 508, 213–235). The analysis has been made over the temperature range ?30°C to 60°C on samples containing 20 to 22% water. At low temperatures the cholestane spin label is tilted with respect to the lipid bilayer normal by an angle of approx. 30° which disappears at the pretransition. In this low temperature range the restricted twisting motion has an activation energy of 5.5 kJ·mol^?1. Above the main transition the twisting motion is unrestricted and has the activation energy 20 kJ·mol^?1. From below the pretransition to above the main transition the velocity of the twisting motion increases by an order of magnitude. The amplitude of the wobbling motion increases abruptly from 0° to 35° at the main transition.     

An electron spin resonance study of cholestane spin label in aqueous mixtures of biliary lipids.

The effect of cholesterol on the fluidity of the phospholipid matrix in mixed micelles derived from bile salts and lecithin has been determined by the paramagnetic probe technique. It was found that correlation times for the cholestane spin label were discontinuous functions of cholesterol content and that these discontinuities correlate with the equilibrium solubility limit for cholesterol in this quaternary system. The origin of these discontinuities is attributed to the existence of another aggregate in addition to the discshaped mixed micelle in lipid solutions supersaturated with cholesterol.     

Localization of dolichols in phospholipid membranes. An ESR spin label study

We have used ESR methods employing spin-labeled stearates to investigate the effects of dolichol on the motion of lipid molecules in phospholipid membranes of phosphatidylethanolamine and phosphatidylcholine. The ESR spectra show that the presence of dolichol affects the motion of the spin probes at carbon-16, but not at carbon-5. Similar results are obtained with phospholipid membranes comprising only phosphatidylcholine. It is suggested that dolichol molecules are present mainly in the lipid core region of phospholipid membranes.     

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.     

Transfer of cholestane spin label between lipid bilayer membranes and its molecular motion in membranes.

The relation between the molecular motion of a steroid in lipid membranes and the transfer rate between membranes was examined using radioactive cholestane spin label. Order parameters of the molecule were determined in bilayers composedof dipalmitoylglycerophosphocholine or egg yolk phosphatidylcholine at various temperatures. The line widths of the ESR signal of the cholestane spin label in membranes, which depend upon the rate of molecular axial rotation in the membranes, were also measured. The temperature dependences of these two parameters and of the transfer rate suggest a close correlation between the rate of molecular axial rotation and the transfer rate.     

Organizational changes in phospholipid multibilayers induced by uncouplers of oxidative phosphorylation: a spin label study

A cholestane spin probe was used to study the effect of uncouplers of oxidative phosphorylation (2,4-dinitrophenol, pentachlorophenol and dicumarol) on the degree of organization of phospholipids in hydrated multibilayers. Disruptive effects were observed—their magnitude depending on pH, time and the presence of cholesterol. A correlation between changes in probe organization and ion conductivity, with maximum effects at the pH corresponding to the pK of the uncoupler, could be demonstrated in the films containing cholesterol. Egg lecithin films containing no cholesterol were disordered maximally at pH 4.0 irrespective of the uncoupler used. The effect of uncouplers on the probe disorganization varied with time after exposure. These time effects indicated that relative movement of uncoupler, probe and lipid molecules occur to produce lipid organizations differing from those after initial exposure to uncoupler. The results show that even in a simple model system uncoupler effects may be complex, and suggest that changes in bilayer lipid organization parameters may play a role in uncoupling oxidative phosphorylation.     

A spin label ESR and saturation transfer ESR study of alpha-tocopherol containing model membranes

An investigation into the effect of alpha-tocopherol on phospholipid model membranes has been carried out by electron spin resonance (ESR) and saturation transfer ESR. The use of stearic acid and of perdeutero -di-t-butyl nitroxide spin probes has allowed us to monitor, in particular, the effect of alpha-tocopherol on both the phospholipid chain order and the phospholipid chain mobility. The results obtained are mainly consistent with a differing action of alpha-tocopherol in the gel and in the liquid crystalline phases: in the former it induces a decrease of order and an increase in fluidity; while in the latter phase an indication of a slight increase in ordering and a clear decrease in fluidity are registered.     

Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: effects of unsaturation and hydration

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852-860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe molecules do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study.

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

ESR spin label studies of the nucleosome core particle and histone core

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label ($\text{3-spiro(2′-(N-}\text{oxyl-4′,4′-dimethyl-oxazolidine))5α-cholestane}$) and a fatty acid spin label ($\text{4′-,4′-dimethyloxazolidine-N-}\text{oxyl}$ derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55°C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron microscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

Protein heat denaturation and study of membrane lipid-protein interactions by spin label ESR.

Spinach chloroplast membranes labelled with stearic acid-spin probe-bearing nitroxyl (label) moiety at 5th, 9th, 12th, 13th, 14th or 16th carbon locations with respect to the carboxylic group of stearic acid were studied (in the dark) by electron spin resonance (ESR) spectroscopy. Spectra were recorded at sample temperatures of 5, 30 and 67 degrees C. After heat denaturation of the membrane proteins for 5 min at 67 degrees C, the spectra were re-recorded at 30 and 5 degrees C for comparison. The results unequivocally show that membrane lipid fatty-acyl chains become substantially more rigid after protein heat-denaturation. The data throw light on the degree of lipid-protein interactions at various microlocations along the length of fatty-acyl chains of the membrane lipid matrix.     

Intra-albumin migration of bound fatty acid probed by spin label ESR

Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.