Can macular xanthophylls replace cholesterol in formation of the liquid-ordered phase in lipid-bilayer membranes?

Lateral organization of membranes made from binary mixtures of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) and macular xanthophylls (lutein or zeaxanthin) was investigated using the saturation-recovery (SR) EPR spin-labeling discrimination by oxygen transport (DOT) method in which the bimolecular collision rate of molecular oxygen with the nitroxide spin label is measured. This work was undertaken to examine whether or not lutein and zeaxanthin, macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity, can parallel other structural functions of cholesterol, including formation of the liquid-ordered phase in membranes. The DOT method permits discrimination of different membrane phases when the collision rates (oxygen transport parameter) differ in these phases. Additionally, membrane phases can be characterized by the oxygen transport parameter in situ without the need for separation, which provides information about the dynamics of each phase. In gel-phase membranes, two coexisting phases were discriminated in the presence of macular xanthophylls - namely, the liquid-ordered-like and solid-ordered-like phases. However, in fluid-phase membranes, xanthophylls only induce the solitary liquid-ordered-like phase, while at similar concentrations, cholesterol induces coexisting liquid-ordered and liquid-disordered phases. No significant differences between the effects of lutein and zeaxanthin were found.     

The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method

Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid- and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (l _o), liquid-disordered (l _d), and solid-ordered (s _o) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the l _o phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the l _d phase, and ∼2 times smaller than in the pure ESM membrane in the s _o phase. The overall change in the oxygen transport parameter is as large as ∼20-fold. Conventional EPR spectra show that 5-SASL is maximally immobilized at the phase boundary between regions with coexisting l _d and l _o phases or s _o and l _o phases and the region with a single l _o phase. The obtained results all _owed for the construction of a phase diagram for the ESM-cholesterol membrane.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 degrees C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with ~5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Tr?uble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9-cis and 13-cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all-trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13-cis and 9-cis zeaxanthin, respectively; whereas, incorporation of all-trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all-trans<9-cis相似文献   

Changes in the plasma membrane of yeast observed by spin label electron spin resonance and caused by photodynamic attack

Spin label electron spin resonance (ESR) was used to characterize the response of lipid regions of the plasma membrane of yeast to photodynamic attack. Following photodynamic attack, the structure of these lipid regions changed resulting in the disappearance of an apparent order—disorder phase transition as well as impeding the diffusion of the steric acid based spin label 12NS into and across the plasma membrane. We propose that singslet molecular oxygen reacting with unsaturated carbon bonds in the fatty acyl chains of lipid surrounding channel proteins leads to an increase in the order of the lipid array and/or a change in the channel protein's conformation and is the cause of the lethal effect of externally sensitized photodynamic action.     

The effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. An electron-electron double resonance (ELDOR) study.

Electron-electron double resonance (ELDOR) and saturation-recovery spectroscopy employing 14N:15N stearic acid spin-label pairs have been used to study the effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. The 14N:15N continuous wave electron-electron double resonance (CW ELDOR) theory has been developed using rate equations based on the relaxation model. The collision frequency between 14N-16 doxyl stearate and 15N-16 doxyl stearate, WHex (16:16), is indicative of lateral diffusion of the spin probes, while the collision frequency between 14N-16 doxyl stearate and 15N-5 doxyl stearate, WHex (16:5), provides information on vertical fluctuations of the 14N-16 doxyl stearate spin probe toward the membrane surface. Our results show that: (a) cholesterol decreases the electron spin-lattice relaxation time Tle of 14N-16 doxyl stearate spin label in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (egg PC). (b) Cholesterol increases the biomolecular collision frequency WHex (16:16) and decreases WHex (16:5), suggesting that incorporation of cholesterol significantly orders the part of the bilayer that it occupies and disorders the interior region of the bilayer. (c) Alkyl chain unsaturation of the host lipid moderates the effect of cholesterol on both vertical fluctuations and lateral diffusion of 14N-16 doxyl stearate. And (d), there are marked differences in the effects of cholesterol on lateral diffusion and vertical fluctuations between 0-30 mol% and 30-50 mol% of cholesterol that suggest an inhomogeneous distribution of cholesterol in the membrane.     

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.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Mapping of collision frequencies for stearic acid spin labels by saturation-recovery electron paramagnetic resonance.

Short pulse saturation-recovery electron paramagnetic resonance methods have been used to measure interactions of 14N:15N stearic acid spin label pairs in multilamellar liposomal dispersions composed of dimyristoyl-phosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC). Pairs consisting of various combinations of [14N]-16-, [14N]-12- or [14N]-5-doxylstearate, and [15N]-16-, [15N]-12-, or [15N]-5-doxylstearate were studied. SR experiments were performed at 27 degrees and 37 degrees C, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer fitting using a damped least-squares approach. The time constants contain combinations of the electron spin lattice relaxation time, Tle, for each member of the spin-label pair, and the Heisenberg exchange rate constant, Kx. Spin-lattice relaxation times for each of the 14N and 15N stearic acid spin labels were determined, and it is noted that Tle for a given 15N-SASL was always slightly greater than that of the corresponding 14N-SASL. From Kx the bimolecular collision frequency was calculated, providing a detailed picture of molecular interactions. For both lipid systems the bimolecular collision rates were ordered as 12:5 less than 16:5 less than 5:5 less than 16:12 less than 12:12 less than 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in DEPC. For the 16:16, 12:12, and 16:12 pairs, Kx was approximately 30% greater in DMPC than in DEPC, a significantly greater difference than is observed by conventional EPR methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spin-label study of the chromaffin granule membrane.

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied. 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32-36 degrees C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 degrees C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature. 3. The lipid-soluble spin label 2,2,6,6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 degrees C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possible involving a re-distribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105-114(1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.