Cytotoxicity of bovine α-lactalbumin: Oleic acid complexes correlates with the disruption of lipid membranes

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V_max, depending on the lipid concentration and vesicle composition, but no change in K_m. Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.     

IFABP portal region insertion during membrane interaction depends on phospholipid composition

Intestinal fatty acid-binding protein (IFABP) is highly expressed in the intestinal epithelium and it belongs to the family of soluble lipid binding proteins. These proteins are thought to participate in most aspects of the biology of lipids, regulating its availability for specific metabolic pathways, targeting and vectorial trafficking of lipids to specific subcellular compartments. The present study is based on the ability of IFABP to interact with phospholipid membranes, and we characterized its immersion into the bilayer's hydrophobic central region occupied by the acyl-chains. We constructed a series of Trp-mutants of IFABP to selectively probe the interaction of different regions of the protein, particularly the elements forming the portal domain that is proposed to regulate the exit and entry of ligands to/from the binding cavity. We employed several fluorescent techniques based on selective quenching induced by soluble or membrane confined agents. The results indicate that the portal region of IFABP penetrates deeply into the phospholipid bilayer, especially when CL-containing vesicles are employed. The orientation of the protein and the degree of penetration were highly dependent on the lipid composition, the superficial net charge and the ionic strength of the medium. These results may be relevant to understand the mechanism of ligand transfer and the specificity responsible for the unique functions of each member of the FABP family.     

Alpha-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains

Previous studies indicate that binding of α-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled α-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that α-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of α-synuclein with membranes in vitro. When compared to wild-type α-synuclein, the disease-causing α-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of α-synuclein with membranes is altered in the inherited forms of Parkinson's disease.     

Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7

Eosinophil cationic protein (ECP/RNase 3) and the skin derived ribonuclease 7 (RNase 7) are members of the RNase A superfamily. RNase 3 is mainly expressed in eosinophils whereas RNase 7 is primarily secreted by keratinocytes. Both proteins present a broad-spectrum antimicrobial activity and their bactericidal mechanism is dependent on their membrane destabilizing capacities. Using phospholipid vesicles as membrane models, we have characterized the protein membrane association process. Confocal microscopy experiments using giant unilamellar vesicles illustrate the morphological changes of the liposome population. By labelling both lipid bilayers and proteins we have monitored the kinetic of the process. The differential protein ability to release the liposome aqueous content was evaluated together with the micellation and aggregation processes. A distinct morphology of the protein/lipid aggregates was visualized by transmission electron microscopy and the proteins overall secondary structure in a lipid microenvironment was assessed by FTIR. Interestingly, for both RNases the membrane interaction events take place in a different behaviour and timing: RNase 3 triggers first the vesicle aggregation, while RNase 7 induces leakage well before the aggregation step. Their distinct mechanism of action at the membrane level may reflect different in vivo antipathogen functions.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two α/β-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating α- and β-amino acid residues, designated simply as α/β-peptide I and α/β-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of α/β-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by α/β-peptide II. The α/β-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of α/β-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. α/β-Peptide II is more effective in this regard because, unlike α/β-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although α/β-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why α/β-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of α/β-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.     

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

The contribution of surface residues to membrane binding and ligand transfer by the α-tocopherol transfer protein (α-TTP)

Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.     

Biophysical Characterization of a New Phospholipid Analogue with a Spin-Labeled Unsaturated Fatty Acyl Chain

Spin-labeled analogs of phospholipids have been used widely to characterize the biophysical properties of membranes. We describe synthesis and application of a new spin-labeled phospholipid analog, SL-POPC. The advantage of this molecule is that the EPR active doxyl group is linked to an unsaturated fatty acyl chain different to saturated phospholipid analogs used so far. The need for those analogs arises from the fact that biological membranes contain unsaturated phospholipids to a large extent. The biophysical properties of SL-POPC in membranes were characterized using EPR and NMR spectroscopy and compared with those of the saturated spin-labeled phospholipid, SL-PSPC. To this end, POPC membranes were labeled with either analog to assess whether the spin-labeled counterpart SL-POPC mimics the membrane properties better than the often used SL-PSPC. The results show that SL-POPC and SL-PSPC explore different molecular environments of the bilayer, and that the type and degree of perturbation of bilayer caused by the label moiety also differs between both analogs. We found that SL-POPC is more appropriate to assess the versatile dynamics of POPC membranes than SL-PSPC.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1

We have studied the binding and interaction of the peptide E1_FP with various model membranes. E1_FP is derived from the amino acid segment 274-291 of the hepatitis C virus envelope glycoprotein E1, which was previously proposed to host the peptide responsible for fusion to target membranes. In the present study we addressed the changes which take place upon E1_FP binding in both the peptide and the phospholipid bilayer, respectively, through a series of complementary experiments. We show that peptide E1_FP binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane and interacts preferentially with cholesterol. The capability of modifying the biophysical properties of model membranes supports its role in HCV-mediated membrane fusion and suggests that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the α-helical part of NPY (amino acids 13-36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1-12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface ΔG supports the preferential interaction of NPY with negatively charged membranes.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.     

Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors but also induce non-opioid excitotoxic effects. Dynorphin A can increase the intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism. In this investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A (1-13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.     

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.     

Properties of Palmitoyl Phosphatidylcholine, Sphingomyelin, and Dihydrosphingomyelin Bilayer Membranes as Reported by Different Fluorescent Reporter Molecules

The properties of vesicle membranes prepared from 16:0-SM, 16:0-DHSM, or DPPC were characterized using steady-state and time-resolved fluorescence spectroscopy and different fluorescent reporter molecules. The acyl-chain region was probed using free and phospholipid-bound 1,6-diphenyl-1,3,5-hexatriene. 16:0-DHSM was found to be the more ordered than both DPPC and 16:0-SM 5°C below and above melting temperature. Interfacial properties of the phospholipid bilayers were examined using 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan), 6-propionyl-2-dimethyl-amino-naphthalene (Prodan), and dansyl-PE. Laurdan and Prodan reported that the two sphingomyelin (SM) membrane interfaces were clearly different from the DPPC membrane interface, whereas the two SM membrane interfaces had more similar properties (both in gel and liquid-crystalline phase). Prodan partition studies showed that membrane resistance to Prodan partitioning increased in the order: 16:0-SM < DPPC < 16:0-DHSM. The degree to which dansyl-PE is exposed to water reflects the structural properties of the membrane-water interface. By comparing the lifetime of dansyl-PE in water and deuterium oxide solution, we could show that the degree to which the dansyl moiety was exposed to water in the membranes increased in the order: 16:0-SM < DPPC < 16:0-DHSM. In conclusion, this study has shown that DHSM forms more ordered bilayers than acyl-chain matched SM or phosphatidylcholine, even in the liquid-crystalline state.