Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Introduction of calcium during hemolysis of erythrocytes causes irreversible membrane changes, including protein aggregation. These changes have been investigated by incorporation of one protein and three fatty acid spin label probes into washed membranes from erythrocytes hemolyzed with a range of Ca2+ concentrations. Electron spin resonance spectra of the lipid probes were analyzed for changes in the order parameters, isotropic coupling constants and mean angular deviations of the lipid hydrocarbon chains. The results generally indicated an increased freedom of mobility of the probes with increased Ca2+ concentration during hemolysis, but the response of each probe showed a different concentration dependence. The maximal response was obtained with the I(5, 10) probe. Variations in the responses were interpreted to reflect different modes of protein-lipid or protein-probe interactions arising from Ca2+ -induced membrane protein alterations. Spectra from membranes treated with the protein spin label showed an increased ratio of immobilized to mobile label with increased Ca2+ concentrations at hemolysis. This is consistent with the membrane protein aggregation phenomena previously observed. It is suggested that the increased protein-protein interactions formed as a result of calcium treatment permit an increased lipid mobility in the membrane regions monitored by the fatty acid probes.     

Estimation of lipid regions in a cytochrome oxidase-lipid complex using spin labeling electron spin resonance: Distribution effects on the spin label

The distribution of lipid in the cytochrome oxidase-lipid complex from beef heart mitochondria has been studied by the spin labeling electron spin resonance technique. The spectra of a phospholipid spin label incorporated in the complex reveals an immobilized (on the ESR time scale) component in addition to the fluid component which is found in aqueous dispersions of the extracted lipids. The first component corresponds to the domain of lipid influenced by the protein, and the second component to the remaining lipid. A theory taking into account not only the sizes of the lipid regions in which the spin label molecule distributes itself, but also the different affinities of the label for the two domains, has been developed. Taking advantage of the variation in spectra obtained with increasing amounts of spin label, computer calculations have been performed to estimate the distribution of lipid in the different regions of the cytochrome oxidase-lipid complex. An extrapolation of the amount of immobilized spin-labeled phospholipid to zero concentration of label allows a calculation of the number of fatty acid residues interacting with the protein to be made. It has been found that the number of aliphatic chains influenced by the protein is higher than that calculated for a single boundary layer around the protein. The approach used in this paper can be useful for studies of protein-lipid interactions in other systems.     

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-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°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 corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°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.     

A spin label study of lipid oxidation catalyzed by heme proteins.

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyoglobin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

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.     

Carbonylcyanide p-trifluoro-methoxyphenylhydrazone-induced change of mitochondrial membrane structure revealed by lipid and protein spin labeling.

Structural information on the phenomena accompanying uncoupling of oxidative phosphorylation in mitochondria was obtained using lipid and protein spin labels. The event of partitioning, observed with a small lipid spin label, the 4,4-dimethyl-2,2-dipentyl-oxazolidine-3-oxide (6-N-11) has been studied. The ratio of polar/hydrophobic part of the third line of the spectra was decreased in the presence of the uncoupler carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP), probably indicating a higher proportion of hydrophobic environment of the label. Protein spin labels have been employed to study mobilities and rate of reduction of the labels. A long-chain maleimide spin label, the 3-2-(2-maleimidoethoxy)ethylcarbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl, in the presence of carbonylcyanide-p-trifluoro-methoxyphenylhydrazone revealed decreases of mobility and of the rate of reduction. Large amplification of these effects was obtained with a short-chain maleimide spin label, the 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. With this spin label, the effect of the uncoupler could be traced down to a concentration of 0.05 μm. It is concluded that both membrane lipid and protein are changed simultaneously in the uncoupling event.     

A new membrane probing steroidal spin label: synthesis and applications

The applicability of a new steroidal spin label, 3-oxo-androstan-17 beta-yl-(2",2",6",6"-tetramethyl-N-oxyl) piperidyl butan-1',4'-dioate, in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline (DPPC) in the presence and absence of drugs has been explored. Its synthesis and characterization has been described herein. Besides, the localization of this spin label in lipid liposomes has been studied using electron spin resonance (ESR), differential scanning calorimetry (DSC) and 1H and 31P NMR spectroscopic techniques. The label has also been used to study the permeability of epinephrine into membrane. The results show that the spin label has a good potential as a spin probe in the study of biomembranes.     

A spin label study of lipid oxidation catalyzed by heme proteins

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyogloblin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

A novel steroidal spin label for membrane structure studies: synthesis and applications

2,2,6,6-Tetramethyl piperidine-N-oxyl nitroxyls are known to partition between aqueous and lipid phases, thus serving as probes to study membrane dynamics. The synthesis of a novel steroidal spin label, 3alpha-hydroxycholan-24-yl-(2",2",6",6"-tetramethyl-N-oxyl)p iperidyl butan-1',4'-dioate, containing 2,2,6,6-tetramethylpiperidine-N-oxyl moiety covalently bonded to the side chain in 3,24-caprostan-diol has been described. The localization of this spin label in model biomembranes has been studied by using electron spin resonance, differential scanning calorimetry, and 1H and 31P NMR spectroscopic techniques. Its applicability in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline in the presence and absence of drugs has been described by using electron spin resonance. The label has also been used to study the permeability of epinephrine into membrane. The results have shown the applicability of the spin label as a potential spin probe in the study of biomembranes.     

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Molecular dynamics simulation of dipalmitoylphosphatidylcholine modified with a MTSL nitroxide spin label in a lipid membrane

We investigate the interaction between dipalmitoylphosphatidylcholine (DPPC) and a nitroxide spin label in order to understand its influences on lipid structure and dynamics using molecular dynamics simulations. The system was modified by covalently attaching nitroxide spin labels to the headgroups of two DPPC molecules. (S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) (MTSL) was used as the spin label. The label position and dynamics were analyzed as was the impact of the modified DPPC on the structure of the surrounding lipids. The modified DPPC molecules locate closer to the center of the membrane than unmodified DPPC molecules. The rotation of the spin label is unrestricted, but there are favored orientations. MTSL depresses the deuterium order parameters of the carbon atoms close to the headgroup in surrounding DPPC molecules. The spin label has no impact on order parameters of carbon atoms at the end of the lipid tails. The lateral diffusion constant of the modified DPPC is indistinguishable from unmodified DPPC molecules. These novel computational results suggest an experimental validation.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling

Transfer of phosphatidylcholine molecules between different membrane fractions of Tetrahymena pyriformis cells grown at 15, 27 and 39.5°C was studied by electron spin resonance (ESR). Microsomes were labeled densely with a phosphatidylcholine spin label and the spin-labeled microsomes were incubated with non-labeled cilia, pellicles or microsomes. The transfer of the phosphatidylcholine spin labels was measured by decrease in the exchange broadening of the electron spin resonance spectrum. In one experiment, the lipid transfer was measured between ³²P-labeled microsomes and non-labeled pellicles by use of their radioactivity. The result was in good agreement with that by ESR. The fluidity of the membrane was estimated using a fatty-acid spin label incorporated into the membranes. Transfer between lipid vesicles was also studied. The results obtained were as follows: (1) The transfer between sonicated vesicles of egg- or dipalmitoyl phosphatidylcholine occurred rapidly in the liquid crystalline phase, with an activation energy of 20 kcal/mol, whereas it hardly occurred in the solid crystalline phase. (2) The transfer rate between microsomal membranes increased with temperature, and an activation energy of the reaction was 17.8 kcal/mol. (3) The transfer from the spin-labeled microsomes to subcellular membranes of the cells grown at 15°C was larger than that to the membranes of the cells grown at 39.5°C. The membrane fluidity was larger for the cells grown at lower temperature. (4) Similar tendency was observed for the transfer between microsomal lipid vesicles prepared from the cells grown at 15°C and at 39.5°C. (5) The transfer from microsomes to various membrane fractions increased in the order, cilia < pellicles < microsomes. The order of increase in the membrane fluidity was cilia < microsomes < pellicles, although the difference between microsomes and pellicles was slight. These results indicate a crucial role of the membrane fluidity in the transfer reaction. (6) Some evidence supported the idea that the lipid transfer between these organelles occurred through the lipid exchange rather than through the fusion.     

Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction

Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.     

250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes.

Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.