Lipid chain length effect on the phase behaviour of PCs/PEG:2000-PEs mixtures. A spin label electron spin resonance and spectrophotometric study

Spin-label electron spin resonance (ESR) spectroscopy and spectrophotometry at fixed wavelength are used to study fully hydrated aqueous dispersions of phosphatidylcholines (PCs) with poly(ethylene glycol:2000)-phosphatidylethanolamines (PEG:2000-PEs). PEG:2000-PE is a micelle-forming polymer-lipid that is extensively used for increasing the lifetime of PC liposomes in the blood circulation through a steric stabilisation effect. The PC lipids and the PEG:2000-PE polymer-lipids have the same acyl chain length of either dimiristoyl (DM) or distearoyl (DS) chains. DMPC/PEG:2000-DMPE and DSPC/PEG:2000-DSPE mixtures were investigated over the entire range of relative compositions (0-100 mol%). In both dispersions, the low-temperature conventional spin label ESR spectra and the temperature dependence of the absorbance at 400 nm give an indication of the conversion from lamellae to micelles with increasing PEG:2000-PEs content. The physical state of the lipid assemblies, lamellar or micellar, is dependent not only on PEG:2000-PEs content, but also on the length of hydrocarbon chain of the lipid matrix. Micellisation is attained more readily in dispersions with longer hydrocarbon chains (i.e. in DSPC/PEG:2000-DSPE mixtures) than in those with shorter acyl chains (i.e. in DMPC/PEG:2000-DMPE mixtures). Saturation transfer ESR (ST-ESR) and absorbance measurements reflect the disaggregation of the bilayers and a reduction in the size of the lipid aggregates by PEG:2000-PEs at low content.     

Sterically stabilized liposomes of DPPC/DPPE-PEG:2000. A spin label ESR and spectrophotometric study

The chain dynamics and the thermotropic phase behavior of sterically stabilized liposomes obtained introducing in the host bilayer matrix of DPPC up to 7 mol% of the polymer-lipid DPPE-PEG:2000 were investigated by spin label electron spin resonance spectroscopy and spectrophotometry. The experimental data indicate that the dispersions have the dynamic and thermotropic characteristics of normal lamellar phase. Moreover, using spin labels that locate both in the interfacial and in the hydrocarbon regions, namely TEMPO-stearate, 5- and 16-PCSL, we find that relative to the unmodified DPPC bilayers, the polymer-grafted bilayers are loosely packed in the interfacial region and have reduced chain mobility in the gel phase. From the temperature dependence of the partition coefficient (P(c)), of the spin probe DTBN between the aqueous and the fluid hydrophobic regions of the bilayers and from the melting curves of the absorbance at 400 nm, we observe a slight influence on the endothermic phase transitions when increasing the concentration of the polymer-lipid in the DPPC bilayers, the influence being more evident in the pre-transition.     

Comparisons of lipid dynamics and packing in fully interdigitated monoarachidoylphosphatidylcholine and non-interdigitated dipalmitoylphosphatidylcholine bilayers: cross polarization/magic angle spinning 13C-NMR studies

13C-NMR spectra have been obtained at 50.3 MHz for monoarachidoylphosphatidylcholine (MAPC) and dipalmitoylphosphatidylcholine (DPPC) dispersions from 25 degrees C to 55 degrees C and for DPPC polycrystals at 25 degrees C using the cross polarization/magic angle spinning technique. Differential scanning calorimetric studies on DPPC and MAPC dispersions show comparable lipid phase transitions with transition temperatures at 41 degrees C and 45 degrees C, respectively, and thus enable the comparison of thermal, structural and dynamic differences between these two systems at corresponding temperatures. Conformational-dependent 13C chemical shift studies on DPPC dispersions demonstrate not only the coexistence of the tilted gel (L beta') and liquid-crystalline (L alpha) phases in the rippled gel (P beta') phase, but also the presence of an intermediate third microscopic phase as evidenced by three resolvable peaks for omega - 1 methylene carbon signals at the temperature interval between Tp and Tm. By comparing chemical shifts of MAPC in the hydrocarbon chain region with those of DPPC at similar reduced temperatures, it can be concluded that the packings are perturbed markedly in the middle segment of the fatty acyl chain during the lamellar to micellar transition. However, terminal methylene and methyl groups of interdigitated MAPC lamellae were found to be more ordered than those of non-interdigitated DPPC bilayers in the gel state. Interestingly, the terminal methyl groups of MAPC in the micelles remain to be relatively ordered; in fact, they are more ordered than the corresponding acyl chain end of DPPC in the liquid-crystalline state. Analysis of data obtained from rotating frame proton spin-lattice relaxation measurements shows a highly mobile phosphocholine headgroup, a rigid carbonyl group and an ordered hydrocarbon chain for lamellar MAPC in the interdigitated state. Furthermore, results suggest that free rotations of the glycerol C2-C3 bond within MAPC molecules may occur in the interdigitated bilayer, whereas intramolecular exchange between two conformations of the glycerol backbone in DPPC become possible at temperatures close to the pretransition temperature. Without isotope enrichment, we conclude that high-resolution solid-state 13C-NMR is indeed a useful technique which can be employed to study the packing and dynamics of phospholipids.     

Kinetics of stearic acid transfer between human serum albumin and sterically stabilized liposomes

The kinetics of the transfer of stearic acids between human serum albumin (HSA) and long circulating sterically stabilised liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of the polymer-lipid poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) have been studied by fluorescence spectroscopy. The study exploits the fact that HSA has a single tryptophan (Trp) residue and that the intrinsic Trp-emission intensity is quenched by the presence of doxyl spin-labelled stearic acids (SASL). Protein/lipid dispersions are considered in which SASL molecules are inserted either in the protein or in the SSL, and the transfer of SASL between the protein and SSL is conveniently monitored by the time variation of the inherent Trp-fluorescence intensity of HSA. It was found that the transfer of fatty acids between HSA and SSL depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers) and on the grafting density of the PEG-lipids at the lipid/protein interface. In the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer, and it is higher for the passage from DPPC bilayers to HSA. The presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer chains, especially at low grafting density and for lipid membranes in the fluid phase.     

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)     

Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes

Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f _p (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.     

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.     

The interfacial structure of phospholipid bilayers: differential scanning calorimetry and Fourier transform infrared spectroscopic studies of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine and its dialkyl and acyl-alkyl analogs.

The thermotropic phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) and its 1,2-dialkyl, 1-acyl 2-alkyl and 1-alkyl 2-acyl analogs was examined by differential scanning calorimetry, and the organization of these molecules in those hydrated bilayers was studied by Fourier transform infrared spectroscopy. The calorimetric data indicate that substitution of either or both of the acyl chains of DPPC with the corresponding ether-linked hydrocarbon chain results in relatively small increases in the temperature (< 4 degrees C) and enthalpy (< 1 kcal/mol) of the lipid chain-melting phase transition. The spectroscopic data reveal that replacement of one or both of the ester-linked hydrocarbon chains of DPPC with its ether-linked analog causes structural changes in the bilayer assembly, which result in an increase in the polarity of the local environments of the phosphate headgroups and of the ester carbonyl groups at the bilayer polar/apolar interface. The latter observation is unexpected, given that ester linkages are considered to be intrinsically more polar that ether linkages. This finding cannot be satisfactorily rationalized unless the conformation of the glycerol backbones of the analogs containing ether-linked hydrocarbon chains differs significantly from that of diacyl glycerolipids such as DPPC. A comparison of the alpha-methylene scissoring bands and the methylene wagging band progressions of these lipids with the corresponding absorption bands of specifically chain-perdeuterated analogs of DPPC also supports the conclusion that replacement of the ester-linked hydrocarbon chains of DPPC with the corresponding ether-linked analog induces conformational changes in the lipid glycerol backbone. The suggestion that the conformation of glycerol backbones in the alkyl-acyl and dialkyl derivatives of DPPC differs from that of the naturally occurring 1,2-diacyl glycerolipid suggests that mono- and di-alkyl glycerolipids may not be good models of their diacyl analogs. These results, and previously published evidence that DPPC analogs with ether-linked hydrocarbon chains spontaneously form chain-interdigitated gel phases at low temperatures, clearly indicate that the properties of lipid bilayers can be substantially altered by small changes in the chemical structures of their polar/polar interfaces, and highlight the critical role of the interfacial region as a determinant of the structure and organization of lipid assemblies.     

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane α-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.     

Raman spectroscopic study of an interdigitated lipid bilayer. Dipalmitoylphosphatidylcholine dispersed in glycerol

Dipalmitoylphosphatidylcholine (DPPC) dispersed in perdeuterated glycerol was investigated in order to determine the effects on the Raman spectra of hydrocarbon chain interdigitation in gel-phase lipid bilayers. Interdigitated DPPC bilayers formed from glycerol dispersions in the gel phase showed a decrease in the peak height intensity $\text{I}{}_{2850}\text{/I}{}_{2880}$ ratio, for the symmetric and asymmetric methylene CH stretching modes, respectively, as compared to non-interdigitated DPPC/water gel-phase dispersions. The decrease in this spectral ratio is interpreted as an increase in chain-chain lateral interactions. Spectra recorded in the 700–740 cm^?1 CN stretching mode region, the 1000–1200 cm^?1 CC stretching mode region and the 1700–1800 cm^? CO stretching mode region were identical for both the interdigitated and non-interdigitated hydrocarbon chain systems. At low temperatures the Raman peak height intensity ratios $\text{I}{}_{2935}\text{/I}{}_{2880}$ were identical for the DPPC/glycerol and DPPC/water dispersions, indicating that this specific index for monitoring bilayer behavior is insensitive to acyl chain interdigitation. The increase, however, in the change of this index at the gel-liquid crystalline phase transition temperature for the DPPC/glycerol dispersions implies a larger entropy of transition in comparison to the non-interdigitated DPPC/water bilayer system.     

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.     

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane alpha-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.     

Cholesterol-induced alterations of the packing properties of gangliosides: an EPR study

The effect of cholesterol (Chol) on two kinds of glycolipid assemblies, one composed of monosialogangliosides (GM1a) and the other formed by a natural mixture of bovine brain gangliosides (TBG), has been analysed. The experimental approach involves spin label electron paramagnetic resonance (EPR) in aqueous lipid dispersions. The employment of a hydrosoluble spin label and a 'quencher' of the EPR signal that is not able to permeate lipid interfaces, allowed us to conclude that GM1a/Chol mixtures give rise to vesicles at Chol proportions for which TBG/Chol mixtures form micelles. The use of different liposoluble spin labels reveals that cholesterol produces a straightening of the hydrocarbon chains in both lipid systems. In GM1a/Chol mixtures, this feature is more pronounced and it is coupled with a decrease in polarity at the chain ends.     

High-resolution electron density profiles reveal influence of fatty acids on bilayer structure.

Small-angle x-ray diffraction studies were performed on gel phase-oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC containing 40 mol% of either palmitic acid (PA) or palmitic acid brominated at the 2-position (BPA). Oriented samples were prepared using a method developed by us, which is as simple as powder sample preparations while offering all the advantages of oriented samples made by traditional methods. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated to further test the consistency of the phase assignments. The diffraction data were used to calculate absolute electron density profiles for different bilayers to a resolution of 5-6 A. Analysis indicates the following: (a) The electron density profiles for the three preparations are virtually identical in the hydrocarbon chain region. (b) There is a decrease in the electron density of the glycerol backbone-headgroup region and d-space in DPPC-PA compared to DPPC. (c) The bromine of fatty-acid brominated at the 2-position is in the vicinity of the glycerol backbone. (d) The bilayer thickness of DPPC containing either brominated or unbrominated fatty acid remains relatively constant with increased levels of hydration, unlike DPPC bilayers.     

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.     

Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model.

Both wide-angle and lamellar x-ray diffraction data are interpreted in terms of a difference in hydrocarbon chain tilt between fully hydrated dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE). Although the hydrocarbon chains of multilayers of DPPC tilt ty approximately 30 degrees relative to the normal to the plane of the bilayer, as previously reported by others, the hydrocarbon chains of DPPE appear to be oriented approximately normal to the plane of the bilayer. It is found that the chain tilt in DPPC bilayers can be reduced by either: (a) adding an n-alkane to the bilayer interiors or (b) adding lanthanum ions to the fluid layers between bilayers. A molecular packing model is presented which accounts for these data. According to this model, DPPC chains tilt because of the size and conformation of the PC polar head group.     

Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules: Application to phospholipid bilayers and interdigitated gel phases

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholines in gel phase lipid bilayers are shown to be sensitive to dipolar spin-spin interactions with paramagnetic ions in the aqueous phase. The reciprocal integrated intensity of the STESR spectrum is linearly dependent on aqueous Ni²⁺ ion concentration, hence, confirming the expectation that the STESR intensity is directly proportional to the spin-lattice relaxation time of the spin label. The gradient of the relaxation rate with respect to Ni²⁺ ion concentration decreases strongly with the position of the nitroxide group down the sn-2 chain of the spin-labeled lipid and is consistent with a 1/R³ dependence on the distance, R, from the bilayer surface. The values derived for the dimensions of the bilayer and lipid molecules in the case of dipalmitoyl phosphatidylcholine (DPPC) are in good agreement with those available from x-ray diffraction studies. Allowance for the multibilayer nature of the DPPC dispersions gives an estimate of the water layer thickness that is also consistent with results from x-ray diffraction. The profile of the paramagnetic ion-induced relaxation is drastically changed with DPPC dispersions in glycerol for which the lipid chains are known to be interdigitated in the gel phase. The terminal methyl groups of the lipid chains are located approximately in register with the C-3 atoms of the sn-2 chain of the oppositely oriented lipid molecules in the interdigitated phase. The thickness of the lipid layer and the effective thickness of the lipid polar group are reduced by ~40% in the interdigitated phase as compared with the bilayer phase. The calibrations of the distance dependence established by use of spin labels at defined chain positions should be applicable to STESR measurements on other biological systems.     

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.     

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.     

Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling

Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB. For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176. The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers. The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB. Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif. For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein. In addition, the collision parameters yielded the position of the protein within the bilayer. The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer. In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel. In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion. The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands. A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.