Interaction of the chemopreventive agent resveratrol and its metabolite,piceatannol, with model membranes

Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer.     

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.     

Headgroup behaviour of an uncharged complex glycolipid

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide—which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.     

Headgroup behaviour of an uncharged complex glycolipid

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide--which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.     

A Glycosphingolipid spin label: Ca2+ effects on sphingolipid distribution in bilayers containing phosphatidyl serine.

A glycosphingolipid (galactosyl ceramide) has been synthesized which has a spin label covalently attached near the methyl end of the fatty acid chain. This is to our knowledge the first glycolipid spin label to be reported. It is being used to study glycosphingolipid behaviour in lipid bilayers — especially with a view to potential differences from phospholipids. Like phospholipids it assumes a random distribution in fluid lipid bilayers but tends to be excluded from regions rich in phosphatidyl serine in the face of a Ca²⁺-induced lateral phase separation.     

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

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

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

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

Association of gangliosides with the lymphocyte plasma membrane studied using radiolabels and spin labels

Gangliosides are known to act as potent suppressors of lectin-stimulated lymphocyte activation when added to the culture medium. Since this effect may be mediated via ganglioside association with (or insertion into) the plasma membrane, we have used 3H- and spin-labelled derivatives of mixed gangliosides to probe the nature of this interaction. Gangliosides bind rapidly to the lymphocyte membrane and show no preference for association with either inside-out or right-side-out membrane vesicles. Around 20% of the bound gangliosides can be removed by repetitive washing, and a further 22-28% by treatment with pronase for 1 h, suggesting that this fraction is tightly bound to membrane proteins at the cell surface. The ESR spectrum of membrane-bound gangliosides did not resemble the spin-exchanged spectrum of micellar spin-labelled gangliosides in aqueous solution, but was similar to that seen for 5 mol% ganglioside spin label in liposomes of egg phosphatidylcholine. This suggests that the bulk of the membrane-bound gangliosides are inserted and molecular dispersed in the lymphocyte membrane. Binding of wheat-germ agglutinin to lymphocyte-associated gangliosides results in specific immobilization of the carbohydrate headgroup, while concanavalin A and other lectins have little or no effect on oligosaccharide mobility. Membrane-inserted gangliosides show a response to lectin binding which is qualitatively different from that seen for gangliosides in bilayers of phosphatidylcholine.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Molecular dynamics simulations of charged and neutral lipid bilayers: treatment of electrostatic interactions

Molecular dynamics (MD) simulations complement experimental methods in studies of the structure and dynamics of lipid bilayers. The choice of algorithms employed in this computational method represents a trade-off between the accuracy and real calculation time. The largest portion of the simulation time is devoted to calculation of long-range electrostatic interactions. To speed-up evaluation of these interactions, various approximations have been used. The most common ones are the truncation of long-range interactions with the use of cut-offs, and the particle-mesh Ewald (PME) method. In this study, several multi-nanosecond cut-off and PME simulations were performed to establish the influence of the simulation protocol on the bilayer properties. Two bilayers were used. One consisted of neutral phosphatidylcholine molecules. The other was a mixed lipid bilayer consisting of neutral phosphatidylethanolamine and negatively charged phosphatidylglycerol molecules. The study shows that the cut-off simulation of a bilayer containing charge molecules generates artefacts; in particular the mobility and order of the charged molecules are vastly different from those determined experimentally. In the PME simulation, the bilayer properties are in general agreement with experimental data. The cut-off simulation of bilayers containing only uncharged molecules does not generate artefacts, nevertheless, the PME simulation gives generally better agreement with experimental data.     

Glycolipid crypticity in membranes--not a simple shielding effect of macromolecules

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their "accessibility'. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed "crypticity' of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.     

Glycolipid crypticity in membranes — not a simple shielding effect of macromolecules

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their ‘accessibility’. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed ‘crypticity’ of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.     

Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c.

Deuterium and phosphorus nuclear magnetic resonance (NMR) has been used to investigate the dynamics of slow motional processes induced in bilayer cardiolipin upon binding with cytochrome c. 31P NMR line shapes suggest that protein binding induces less restricted, isotropic-like motions in the lipid phosphates within the ms time scale of this measurement. However, these motions impart rapid transverse relaxation to methylene deuterons adjacent to the phosphate in the lipid headgroup and so did not feature strongly in the NMR line shapes recorded from these nuclei by using the quadrupolar echo. Nonetheless, motional characteristics of the headgroup deuterons were accessible to a dynamic NMR approach using the Carr-Purcell-Meiboom-Gill multiple-pulse experiment. Compared to the well-studied case of deuterons in fatty acyl chains of bilayer phosphatidylcholine, the motions determining the 2H spin transverse relaxation in the headgroup of bilayer cardiolipin were much faster, having a lower limit in the 5-10 kHz range. On binding with cytochrome c, the T2 effecting motions in the cardiolipin headgroup became faster still, with rates comparable to the residual quadrupolar coupling frequency of the headgroup deuterons (approximately 25 kHz) and so coincided with the time scale for recording the quadrupolar echo (approximately 40 microseconds). It is concluded that the headgroup of cardiolipin does not exclusively report localized dynamic information but is particularly sensitive to collective motions occurring throughout the bilayer molecules. Although the rates of collective modes of motion may be dependent on the lipid type in pure lipid bilayers, these low-frequency fluctuations appear to occupy a similar dynamic range in a variety of lipid-protein systems, including the natural membranes.     

Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum Ca2+-ATPase

A multiple equilibrium binding model is used to examine phospholipid and cholesterol binding with the transmembranous protein Ca2+-ATPase (calcium pump). The protein was reconstituted in egg phosphatidylcholine bilayers by lipid substitution of rabbit muscle sarcoplasmic reticulum. Electron spin resonance spectra of a phosphatidylcholine spin-label and a recently developed cholesterol spin-label show two major spectral contributions, a motionally restricted component consistent with interactions between the label and the protein surface and another component characteristic of motion of the label in a fluid lipid bilayer. The number of lipid binding (or contact) sites at the hydrophobic surface of the protein is calculated to be N = 22 +/- 2. Experiments with intact sarcoplasmic reticulum membranes give approximately the same value for N. The relative binding constants are Kav approximately 1 for the phosphatidylcholine label and Kav approximately 0.65 for the cholesterol spin-label. Thus, cholesterol does contact the surface of the protein, but with a somewhat lower probability than phosphatidylcholine. This is confirmed by competition experiments where unlabeled cholesterol and the phospholipid spin-label are both present in the bilayer. Evidently the flexible acyl chains of the phospholipid molecules accommodate more readily to the irregular surface of the protein than does the rigid steroid structure of cholesterol.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore’s immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.     

Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon

'Interdigitation' is a term coined to describe the phenomenon whereby pure phosphatidylcholines with intramolecular fatty acid chain length heterogeneity when hydrated to form bilayers may insert the methyl ends of long fatty acids from one side across more than half of the membrane thickness to protrude amongst the acyl chains of the opposite side of the bilayer (Keough, K.M.W. and Davis, P.J. (1979) Biochemistry 18, 1453-1459; Huang, C. and Mason, J.T. (1986) Biochim. Biophys. Acta 864, 423-470). In this article we address the fate of long fatty acid chains of glycosphingolipids present as minor components in membranes of non-interdigitating phosphatidylcholines. In this pursuit, derivatives of galactosyl ceramide, lactosyl ceramide, globoside and GM1 were synthesized having either 18-carbon or 24-carbon fatty acid with a spin label covalently attached at C-16. Labelled glycolipids were incorporated at 1-2 mol% into bilayers of synthetic phosphatidylcholines, their mixtures with cholesterol, or natural egg phosphatidylcholine. In each case the C-16 carbon of the glycolipid long chain fatty acid showed considerably greater 'order' and immobility than did C-16 of the fatty acid which was similar in length to the host matrix phospholipids. We interpret this as strong evidence that the long chain fatty acid interdigitates across the mid point of the bilayer in the systems studied. Clearly this phenomenon did not require that the phospholipid host matrix have mixed chain lengths. Furthermore it was totally independent of glycolipid family: for a given host matrix and (glycolipid) fatty acid chain length the order parameter values found were the same amongst all four glycolipid families tested.     

Lipid protein interactions in mitochondria. Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes.

The effect of n-butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n-alcohols is related to their hydrophobicity.Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data.As for the spin label data, the effect of the series of aliphatic n-alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.     

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Deltanu, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.     

Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.