Dynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state.

The effects of cholesterol on the dynamics of cholestane spin probe (CSL) in various phosphatidylcholine-cholesterol mixed model membranes are examined. The lateral diffusion, D of CSL in DMPC/POPC/cholesterol ternary mixtures, is measured utilizing an improved dynamic imaging electron spin resonance method. It shows a factor of two decrease at 10 mol % and 25 degrees C, whereas it shows only a 40% decrease at 50 mol % and 50 degrees C. A comparison with results in POPC/cholesterol mixtures, which show a stronger effect of cholesterol on D, indicates that acyl chain unsaturation leads to stronger self association of cholesterol in PC model membranes. An S2CSL dependence of the activation energy for D, has been confirmed for the DMPC/POPC/cholesterol mixtures. Here SCSL is the order parameter for CSL. A similar correlation of R perpendicular, the perpendicular component of the rotational diffusion coefficient, with SCSL, which is true for all three mixtures (DMPC/cholesterol, POPC/cholesterol, and DMPC/POPC/cholesterol) we have studied, is also found. These are associated with the effects of enhanced local ordering on the free volume needed for translation and reorientation. Such correlations of dynamic properties D and R perpendicular with the thermodynamic quantity S, as well as the consistent interpretations of the effect of acyl chain unsaturation on the dynamics in terms of the activity coefficients, strongly emphasize the interrelation between the dynamic structure and the thermodynamics of the PC/cholesterol mixtures.     

Dynamic imaging of lateral diffusion by electron spin resonance and study of rotational dynamics in model membranes. Effect of cholesterol.

The effects of cholesterol on the dynamics and the structural properties of two different spin probes, the sterol type CSL and the phospholipid type 16-PC, in POPC/cholesterol oriented multilayer model membranes were examined. Our results are consistent with a nonideal solution containing cholesterol-rich clusters created by the self association of cholesterol in POPC model membranes. The lateral diffusion coefficient D of the spin probes was measured over the temperature range of 15 to 60 degrees C and over the concentration range of 0 to 30 mol% of cholesterol in the model membrane by the electron spin resonance (ESR) imaging method. The rotational diffusion coefficients (including R perpendicular) and the order parameter S were determined utilizing a nonlinear least square ESR spectral simulation method. D, R perpendicular and S of CSL deviate considerably from linear dependence on mole percent cholesterol. The D of CSL was decreased by a factor of four at 15 degrees C and a factor of two at 60 degrees C for concentrations of cholesterol over 10 mol %, whereas those of 16-PC were hardly affected. Cholesterol decreased R perpendicular by a factor of 10 at 30 mol % of cholesterol, but it increased slightly that of 16-PC. A significant increase of S for CSL due to the presence of cholesterol was observed. It is shown how the difference in variation of S for CSL vs. 16-PC with composition may be interpreted in terms of their respective activity coefficients, and how a single universal linear relation is obtained for the S of both probes in terms of a scaled temperature. Simple but general correlations of D and of R perpendicular with S were also found, which aid in the interpretation of these diffusion coefficients.     

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

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

Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance

The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.     

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.     

Studies on lipid membranes by two-dimensional Fourier transform ESR: Enhancement of resolution to ordering and dynamics.

The first two-dimensional Fourier-transform electron spin resonance (2D-FT-ESR) studies of nitroxide-labeled lipids in membrane vesicles are reported. The considerable enhancement this experiment provides for extracting rotational and translational diffusion rates, as well as orientational ordering parameters by means of ESR spectroscopy, is demonstrated. The 2D spectral analysis is achieved using theoretical simulations that are fit to experiments by an efficient and automated nonlinear least squares approach. These methods are applied to dispersions of 1-palmitoyl-2oleoyl-sn-glycerophosphatidylcholine (POPC) model membranes utilizing spin labels 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidylcholine and the 3-doxyl derivative of cholestan-3-one (CSL). Generally favorable agreement is obtained between the results obtained by 2D-FT-ESR on vesicles with the previous results on similar systems studied by continuous wave (cw) ESR on aligned samples. The precision in determining the dynamic and ordering parameters is significantly better for 2D-FT-ESR, even though the cw ESR spectra from membrane vesicles are resolved more poorly than those from well aligned samples. Some small differences in results by the two methods are discussed in terms of limitations of the methods and/or theoretical models, as well as possible differences between dynamic molecular structure in vesicles versus aligned membranes. An interesting observation with CSL/POPC, that the apparent homogeneous linewidths seem to increase in "real time," is tentatively attributed to the effects of slow director fluctuations in the membrane vesicles.     

Reorientational dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation

Electron spin resonance experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in multilamellar liposomes and small unilamellar vesicles (SUVs) of palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). The experimental spectra were analyzed by a numerical solution of the stochastic Liouville equation. Effects of temperature, presence of unsaturated bonds and high bilayer curvature on the dynamic behaviour of the lipid molecules were studied. Our results, combined with results from planar multibilayers with a varying hydration rate (Korstanje et al. (1989) Biochim. Biophys. Acta 980, 225-233), give a consistent picture of the orientational order and rotational dynamics of CSL molecules embedded in lipid matrices with various geometrical configurations. Increase of hydration or temperature reduces molecular ordering and increases molecular dynamics. In highly curved vesicle configurations, SUVs, molecular order is found to be lower than in multilamellar liposomes. In contrast, rotational motion is not affected by increase of curvature. In all lipid configurations studied, increase of the number of unsaturated bonds in the fatty acid chains reduces molecular ordering. We find, however, no effect of unsaturation on the rotational mobility of the CSL probe molecules. These results clearly show that changes in molecular orientational order and reorientational dynamics have to be considered separately, and that they are not necessarily correlated as implied by the common concept of membrane fluidity. Comparing our results with data from a motional narrowing analysis shows that the latter approach seriously overestimates the rate of molecular reorientation.     

An electron spin resonance study of interactions between gramicidin A'' and phosphatidylcholine bilayers.

The model of microscopic order and macroscopic disorder was used to stimulate electron spin resonance spectra of spin-labeled lipids, 5-PC, 10-PC, and 16-PC in multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) containing gramicidin A' (GA) at temperatures above the gel-to-liquid crystal transition of DPPC. The simulations show that at a lower concentration of GA (i.e., molar ratios of DPPC/GA greater than 3), GA has only a slight effect on the acyl chain dynamics. The rotational diffusion rate around the axis parallel to the long hydrocarbon chain remains unchanged or increases slightly, while the rate around the perpendicular axes decreases slightly. These spectra from DPPC/GA mixtures could only be fit successfully with two or more components consistent with the well-known concept of "boundary lipids," that is, the peptide induces structural inhomogeneity in lipid bilayers. However, the spectra were significantly better fit with additional components that exhibit increased local ordering, implying decreased amplitude of rotational motion, rather than immobilized components with sharply a reduced rotational rate. The largest relative effects occur at the end of the acyl chains, where the average local order parameter St of 16-PC increases from 0.06 for pure lipid to 0.66 for 1:1 DPPC/GA. The inhomogeneity in ordering in DPPC bilayers due to GA decreases with increasing temperature. The hyperfine tensor component Azz increases for 10-PC and 16-PC when GA is incorporated into DPPC bilayers, indicating that water has deeply penetrated into the DPPC bilayers. Simulations of published electron spin resonance spectra of 14-PC in dimyristoylphosphatidylcholine/cytochrome oxidase complexes were also better fit by additional components that were more ordered, rather than immobilized. The average local order parameter in this case is found to increase from 0.11 for pure dimyristoylphosphatidylcholine to 0.61 for a lipid/protein ratio of 50. These spectra and their simulations are similar to the results obtained with 16-PC in the DPPC/GA mixtures. The relevance to studies of lipid-protein interactions for other proteins is briefly discussed.     

A 2D-ELDOR study of the liquid ordered phase in multilamellar vesicle membranes

2D-ELDOR spectroscopy has been employed to study the dynamic structure of the liquid-ordered (Lo) phase versus that of the liquid-crystalline (Lc) phase in multibilayer phospholipid vesicles without (Lc) and with (Lo) cholesterol, using end-chain and headgroup labels and spin-labeled cholestane. The spectra are in most cases found to be dramatically different for these two phases. Thus, visual inspection of the 2D-ELDOR spectra provides a convenient way to distinguish the two phases in membranes. Detailed analysis shows these observations are due to increased ordering in the Lo phase and modified reorientation rates. In the Lo phase, acyl chains undergo a faster rotational diffusion and higher ordering than in the Lc phase, whereas spin-labeled cholestane exhibits slower rotational diffusion and higher ordering. On the other hand, the choline headgroup in the Lo phase exhibits faster motion and reduced but realigned ordering versus the Lc phase. The microscopic translational diffusion rates in the Lo phase are significantly reduced in the presence of cholesterol. These results are compared with previous studies, and a consistent model is provided for interpreting them in terms of the differences in the dynamic structure of the Lo and Lc phases.     

Insight into the putative specific interactions between cholesterol, sphingomyelin, and palmitoyl-oleoyl phosphatidylcholine

The effects of cholesterol (Chol) on phospholipid bilayers include ordering of the fatty acyl chains, condensing of the lipids in the bilayer plane, and promotion of the liquid-ordered phase. These effects depend on the type of phospholipids in the bilayer and are determined by the nature of the underlying molecular interactions. As for Chol, it has been shown to interact more favorably with sphingomyelin than with most phosphatidylcholines, which in given circumstances leads to formation of lateral domains. However, the exact origin and nature of Chol-phospholipid interactions have recently been subjects of speculation. We examine interactions between Chol, palmitoylsphingomyelin (PSM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in hydrated lipid bilayers by extensive atom-scale molecular dynamics simulations. We employ a tailored lipid configuration: Individual PSM and Chol monomers, as well as PSM-Chol dimers, are embedded in a POPC lipid bilayer in the liquid crystalline phase. Such a setup allows direct comparison of dimeric and monomeric PSMs and Chol, which ultimately shows how the small differences in PSM and POPC structure can lead to profoundly different interactions with Chol. Our analysis shows that direct hydrogen bonding between PSM and Chol does not provide an adequate explanation for their putative specific interaction. Rather, a combination of charge-pairing, hydrophobic, and van der Waals interactions leads to a lower tilt in PSM neighboring Chol than in Chol with only POPC neighbors. This implies improved Chol-induced ordering of PSM's chains over POPC's chains. These findings are discussed in the context of the hydrophobic mismatch concept suggested recently.     

Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.     

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.     

An AFM study of solid-phase bilayers of unsaturated PC lipids and the lateral distribution of the transmembrane model peptide WALP23 in these bilayers

An altered lipid packing can have a large influence on the properties of the membrane and the lateral distribution of proteins and/or peptides that are associated with the bilayer. Here, it is shown by contact-mode atomic force microscopy that the surface topography of solid-phase bilayers of PC lipids with an unsaturated cis bond in their acyl chains shows surfaces with a large number of line-type packing defects, in contrast to the much smoother surfaces observed for saturated PC lipids. Di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC (POPC) were used. Next, the influence of an altered lipid environment on the lateral distribution of the single α-helical model peptide WALP23 was studied by incorporating the peptide in the bilayers of di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC unsaturated lipids. The presence of WALP23 leads to an increase in the number of packing defects but does not lead to the formation of the striated domains that were previously observed in bilayers of saturated PC lipids and WALP. This is ascribed to the less efficient lateral lipid packing of the unsaturated lipids, while the increase in packing defects is probably an indirect effect of the peptide. Finally, the fact that an altered lipid packing affects the distribution of WALP23 is also confirmed in an additional experiment where the solvent TFE (2,2,2-trifluorethanol) is added to bilayers of di-16:0-PC/WALP23. At 3.5 vol% TFE, the previous striated ordering of the peptide is abolished and replaced by loose lines.     

PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study

PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids. We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserines. For this purpose, mixed bilayers of 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes (POPC/POPS 5:1, m/m). Spectra of headgroup- and chain-deuterated POPC and POPS phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at different temperatures and for various concentrations of the PMP1 fragment. Data obtained from POPS deuterons revealed the formation of specific peptide-POPS complexes giving rise to a slow exchange between free and bound PS lipids, scarcely observed in solid-state NMR studies of lipid-peptide/protein interactions. The stoichiometry of the complex (8 POPS per peptide) was determined and its significance is discussed. The data obtained with headgroup-deuterated POPC were rationalized with a model that integrates the electrostatic perturbation induced by the cationic peptide on the negatively charged membrane interface, and a "spacer" effect due to the intercalation of POPS/PMP1f complexes between choline headgroups.     

Structural determinants of miscibility in surface films of galactosylceramide and phosphatidylcholine: effect of unsaturation in the galactosylceramide acyl chain

The Langmuir film balance technique has been used to define the surface structure and determine the mixing behavior of galactosylceramide (GalCer) and phosphatidylcholines in surface phases. To determine the effect of unsaturation on surface behavior, chain-pure GalCer species containing either oleoyl (18:1 delta 9), eicosenoyl (20:1 delta 11), or eicosadienoyl (20:2 delta 11,14) fatty acyl chains were synthesized. Using bovine brain GalCer as a reference, surface pressure versus molecular area (phi-A) isotherms of the pure lipids were measured and analyzed by determining their compressibilities and by using an equation of state for lipid monolayers. This information, when coupled with surface potential versus molecular area (delta V-A) analyses, provides insights into GalCer surface structure in terms of molecular packing and orientation. Lipid mixing behavior was determined by classical approaches which involve analyzing the average molecular area, the average surface dipole moment, and surface pressure as a function of film composition. The results indicate that, in contrast to the complex mixing behavior displayed by bovine brain GalCer and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), chain-pure GalCer species containing either oleoyl, eicosenoyl, or eicosadienoyl fatty acyl chains are miscible with POPC over the entire composition range. Moreover, increasing amounts of GalCer containing eicosenoyl acyl chains systematically elevate dipalmitoylphosphatidylcholine's (DPPC) liquid-expanded-to-liquid-condensed transition pressure. Such behavior is consistent with GalCer being miscible with the liquid-expanded phase of DPPC. Thus, fatty acyl unsaturation is a critical parameter governing the mixing behavior of GalCer with phosphatidylcholine.     

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.     

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

Lipid-gramicidin interactions: dynamic structure of the boundary lipid by 2D-ELDOR

The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.