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Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events as they form glycolipid-rich membrane domains, such as lipid rafts. GSLs consist of a certain oligosaccharide head group and a ceramide tail with various lengths of acyl chains. The structure of the head group as well as the carbon number and degree of the unsaturation of the acyl chain are known to regulate the membrane distributions and interleaflet couplings of GSLs by altering physicochemical properties, such as dynamics, interactions, and cluster sizes. This chapter provides the detailed use of time-resolved fluorescence measurement for investigating the membrane properties of lactosylceramide (LacCer)-enriched domains in bilayer membranes. LacCer belongs to the neutral GSLs and is believed in forming a highly ordered phase in model membranes and biological membranes, while the details of the domain remain unclear. Here, we suggest using trans-parinaric acid (tPA) and tPA-LacCer fluorescent probes to reveal the dynamics and size of the GSL domains since they prefer to be distributed in the GSL-rich ordered phase. The fluorescence lifetime in the nanosecond timescale reveals the difference in the surrounding membrane environments, which relates to hydrocarbon chain ordering, membrane hydration, and submicrometer domain size. The fluorescence lifetime of these probes can thus provide important information on submicron- to nano-scale small GSL domains not only in model membranes but also in biological membranes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane., (© 2022. The Author(s).)

The chain melting of lipid bilayers has often been investigated in detail using calorimetric methods, such as differential scanning calorimetry (DSC), and the resultant main transition temperature is regarded as one of the most important parameters in model membrane experiments. However, it is not always clear whether the hydrocarbon chains of lipids are gradually melting along the depth of the lipid bilayer or whether they all melt concurrently in a very narrow temperature range, as implied by DSC. In this study, we focused on stearoyl-d-sphingomyelin (SSM) as an example of raft-forming lipids. We synthesized deuterium-labeled SSMs at the 4', 10', and 16' positions, and their depth-dependent melting was measured using solid-state deuterium NMR by changing the temperature by 1.0 °C, and comparing with that observed from a saturated lipid, palmitoylstearoylphosphatidylcholine (PSPC). The results showed that SSM exhibited a characteristic depth-dependent melting, which was not observed for PSPC. The strong intermolecular hydrogen bonds between the sphingomyelin amide moiety probably caused the chain melting to start from the chain terminus through the middle part and end in the upper part. This depth-dependent melting implies that the small gel-like domains of SSM remain at temperatures slightly above the main transition temperature. These sphingomyelin features may be responsible for the biological properties of SM-based lipid rafts.

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the 2 H NMR spectra of 10',10'-d 2 -LacCer and 18',18',18'-d 3 -LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ceramides can regulate biological processes probably through the formation of laterally segregated and highly packed ceramide-rich domains in lipid bilayers. In the course of preparation of its analogues, we found that a hydrogen-bond-competent functional group in the C1 position is necessary to form ceramide-rich domains in lipid bilayers [Matsufuji; Langmuir 2018]. Hence, in the present study, we newly synthesized three ceramide analogues: CerN 3 , CerNH 2 , and CerNHAc, in which the 1-OH group of ceramide is substituted with a nitrogen functionality. CerNH 2 and CerNHAc are capable of forming hydrogen bonds in their headgroups, whereas CerN 3 is not. Fluorescent microscopy observation and differential scanning calorimetry analysis disclosed that these ceramide analogues formed ceramide-rich phases in sphingomyelin bilayers, although their thermal stability was slightly inferior to that of normal ceramides. Moreover, wide-angle X-ray diffraction analysis showed that the chain packing structure of ceramide-rich phases of CerNHAc and CerN 3 was similar to that of normal ceramide, while the CerNH 2 -rich phase showed a slightly looser chain packing due to the formation of CerNH 3 + . Although the domain formation of CerN 3 was unexpected because of the lack of hydrogen-bond capability in the headgroup, it may become a promising tool for investigating the mechanistic link between the ceramide-rich phase and the ceramide-related biological functions owing to its Raman activity and applicability to click chemistry.

Actinoporins constitute a family of α pore-forming toxins produced by sea anemones. The soluble fold of these proteins consists of a β-sandwich flanked by two α-helices. Actinoporins exert their activity by specifically recognizing sphingomyelin at their target membranes. Once there, they penetrate the membrane with their N-terminal α-helices, a process that leads to the formation of cation-selective pores. These pores kill the target cells by provoking an osmotic shock on them. In this review, we examine the role and relevance of the structural features of actinoporins, down to the residue level. We look at the specific amino acids that play significant roles in the function of actinoporins and their fold. Particular emphasis is given to those residues that display a high degree of conservation across the actinoporin sequences known to date. In light of the latest findings in the field, the membrane requirements for pore formation, the effect of lipid composition, and the process of pore formation are also discussed., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores.

Protein-lipid interactions are crucial events from a biochemical point of view, like the interaction of proteins with the cell plasma membrane, and their study is of great importance. Actinoporins are a very powerful tool to study this kind of interactions, since they are soluble proteins in an aqueous environment, capable of inserting into membranes when they have the adequate composition. In fact, actinoporins have been used to study protein-lipid interactions for many years now. Sometimes it is not possible to use real biological membranes in the experiments, so model membranes need to be used. This article aims to give a thorough description of many of the techniques used to study actinoporin-lipid interactions, using both biological and model membranes: Hemolysis, release of vesicles content, surface plasmon resonance, isothermal titration calorimetry, fluorescence-based measurements, etc. Some of these techniques measure the actinoporins activity and some measure their binding properties. The combination of all the techniques described can offer valuable information about the thermodynamics and the kinetics of the actinoporin-lipid interaction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey-predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus . Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.

Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades. However, regardless of the probe of choice, the results reported that StnII action was never able to empty the vesicles completely. This was hard to explain if StnII pores were to be stable and always leaky for the probes used. To address this question, we have used a variety of probes, including rhodamine 6G or Tb 3+ , to test the permeability of StnII's pores. Our results indicate that calcein was in fact too large to fit through StnII's pores, and that the standard method in the field is actually reporting StnII-induced transient permeation of the membrane rather than the passage of solutes through the stable assembled pores. In order to evaluate the permeability of these structures, we used a dithionite-based assay, which showed that the final pores were in fact open. Thus, our results indicate that the stable actinoporins' pores are open in spite of plateaued classic release curves. Besides the proper pore, the first stages of pore formation would inflict serious damage to living cells as well., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII's spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of β-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sphingomyelin (SM), a major component of small domains (or lipid rafts) in mammalian cell membranes, forms a liquid-ordered phase in the presence of cholesterol (Cho). However, the nature of molecular interactions within the ordered SM/Cho phase remains elusive. We previously revealed that stearoyl-SM (SSM) and its enantiomer (ent-SSM) separately form nano-subdomains within the liquid-ordered phase involving homophilic SSM-SSM and ent-SSM-ent-SSM interactions. In this study, the details of the subdomain formation by SSMs at the nanometer range were examined using Förster resonance energy transfer (FRET) measurements in lipid bilayers containing SSM and ent-SSM, dioleoyl-phosphatidylcholine and Cho. Although microscopy detected a stereochemical effect on partition coefficient favoring stereochemically homophilic interactions in the liquid-ordered state, it showed no significant difference in large-scale liquid-ordered domain formation by the two stereoisomers. In contrast to the uniform domains seen microscopy, FRET analysis using fluorescent donor- and acceptor-labeled SSM showed distinct differences in SM and ent-SM colocalization within nanoscale distances. Donor- and acceptor-labeled SSM showed significantly higher FRET efficiency than did donor-labeled SSM and acceptor-labeled ent-SSM in lipid vesicles composed of "racemic" (1:1) mixtures of SSM/ent-SSM with dioleoylphosphatidylcholine and Cho. The difference in FRET efficiency indicated that SSM and ent-SSM assemble to form separate nano-subdomains. The average size of the subdomains decreased as temperature increased, and at physiological temperatures, the subdomains were found to have a single-digit nanometer radius. These results suggest that (even in the absence of ent-SM) SM-SM interactions play a crucial role in forming nano-subdomains within liquid-ordered domains and may be a key feature of lipid microdomains (or rafts) in biological membranes., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Lateral segregation and the formation of lateral domains are well-known phenomena in ternary lipid bilayers composed of an unsaturated (low gel-to-liquid phase transition temperature (T m )) phospholipid, a saturated (high-T m ) phospholipid, and cholesterol. The formation of lateral domains has been shown to be influenced by differences in phospholipid acyl chain unsaturation and length. Recently, we also showed that differential interactions of cholesterol with low- and high-T m phospholipids in the bilayer can facilitate phospholipid segregation. Now, we have investigated phospholipid-cholesterol interactions and their role in lateral segregation in ternary bilayers composed of different unsaturated phosphatidylcholines (PCs) with varying acyl chain lengths, N-palmitoyl-D-erythro-sphingomyelin (PSM), and cholesterol. Using deuterium NMR spectroscopy, we determined how PSM was influenced by the acyl chain composition in surrounding PC environments and correlated this with the affinity of cholestatrienol (a fluorescent cholesterol analog) for PSM in the different PC environments. Results from a combination of time-resolved fluorescence measurements of trans-parinaric acid and Förster resonance energy transfer experiments showed that the relative affinity of cholesterol for phospholipids determined the degree to which the sterol promoted domain formation. From Förster resonance energy transfer, deuterium NMR, and differential scanning calorimetry results, it was clear that cholesterol also influenced both the thermostability of the domains and the degree of order in and outside the PSM-rich domains. The results of this study have shown that the affinity of cholesterol for both low-T m and high-T m phospholipids and the effects of low- and high-T m phospholipids on each other influence both lateral structure and domain properties in complex bilayers. We envision that similar effects also contribute to lateral heterogeneity in even more complex biological membranes., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Lateral segregation of lipids in model and biological membranes has been studied intensively in the last decades using a comprehensive set of experimental techniques. Most methods require a probe to report on the biophysical properties of a specific molecule in the lipid bilayer. Because such probes can adversely affect the results of the measurement and perturb the local membrane structure and dynamics, a detailed understanding of probe behavior and its influence on the properties of its direct environment is important. Membrane phase-selective and lipid-mimicking molecules represent common types of probes. Here, we have studied how the fluorescent probes trans -parinaric acid (tPA), diphenylhexatriene (DPH), and 1-oleoyl-2-propionyl[DPH]- sn -glycero-3-phosphocholine (O-DPH-PC) affect the membrane properties of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers using 2 H and 31 P NMR spectroscopy in the solid state. In addition, using 2D 1 H magic-angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) NMR, we have determined the distribution of the probe moieties in the POPC membrane parallel to the membrane normal. We found that the different probes exhibit distinct membrane localizations and distributions, e.g . tPA is located parallel to the membrane normal while DPH predominantly exist in two orientations. Further, tPA was conjugated to sphingomyelin (tPA-SM) as a substitute for the acyl chain in the SM. 1 H NOESY NMR was used to probe the interaction of the tPA-SM with cholesterol as dominant in liquid ordered membrane domains in comparison to POPC-cholesterol interaction in membranes composed of ternary lipid mixtures. We could show that tPA-SM exhibited a strong favorable and very temperature-dependent interaction with cholesterol in comparison to POPC. In conclusion, the NMR techniques can explain probe behavior but also be used to measure lipid-specific affinities between different lipid segments and individual molecules in complex bilayers, relevant to understanding nanodomain formation in biological membranes.

Ceramide-1-phosphate is a minor sphingolipid with important functions in cell signaling. In this study, we examined the propensity of palmitoyl ceramide-1-phosphate (Cer-1P) to segregate laterally into ordered domains in different bilayer compositions at 23 and 37°C and compared this with segregation of palmitoyl ceramide (PCer) and palmitoyl sphingomyelin (PSM). The ordered-domain formation in the fluid phosphatidylcholine bilayers was determined using the emission lifetime changes of trans-parinaric acid and from differential scanning calorimetry thermograms. The lateral segregation of Cer-1P was examined when hydrated to bilayers in Tris buffer (50 mM Tris, 140 mM NaCl (pH 7.4)). At this pH, Cer-1P was negatively charged. The lateral segregation propensity of Cer-1P in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers was intermediate between PCer and PSM. Based on differential scanning calorimetry analysis, we observed that the gel domains formed by Cer-1P in POPC bilayers (POPC:Cer-1P 70:30 by mol) were less stable (melting interval 16-37°C) than the corresponding POPC and PCer gel domains at equal composition (melting interval 20-55°C). The gel-phase melting enthalpy was also much lower in Cer-1P (1.5 kcal/mol) than in the PCer-containing POPC bilayers (9 kcal/mol). Cer-1P appeared to be at least partially miscible with PCer domains in POPC bilayers. Cer-1P domains were stabilized in the presence of PSM (POPC:PSM 85:15), similarly as seen with PCer-rich domains. In bilayers at 37°C, with an approximate outer-leaflet cell membrane composition (sphingomyelin and cholesterol enriched, aminophospholipid poor), Cer-1P segregation did not lead to the formation of ordered domains, at least when compared with PCer segregation. In bilayers with an approximate inner-leaflet composition (sphingomyelin poor, cholesterol and aminophospholipid enriched), Cer-1P also failed to form ordered domains. PCer segregated into ordered domains only after the PCer/cholesterol ratio exceeded an approximate equimolar ratio., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp) mutants to study StnII/bilayer interactions. First, we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done because both Chol and OCer have a 1-hydroxyl, which helps to orient the molecule in the bilayer (although OCer has additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization; OCer failed to activate it. To further examine possible Chol/StnII interactions, we measured Förster resonance energy transfer between Trp in StnII and cholestatrienol, a fluorescent analog of Chol. We could show higher Förster resonance energy transfer efficiency between cholestatrienol and Trps in position 100 and 114 of StnII when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The hydration states of the interfacial region of lipid bilayers were investigated on the basis of the time-resolved emission spectra (TRES) analysis of 6-lauroyl-2-dimethylamino naphthalene (Laurdan), a common fluorescence probe used to analyze membrane hydration. TRES derived from long and short lifetime components were extracted from samples of different lipid species: 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), d- erythro- N-palmitoyl-sphingosylphosphorylcholine (PSM), and a DOPC/PSM binary bilayer system. Neither lifetime component (short or long) corresponded with the hydration properties; the short lifetime component of DOPC (1.97 ns) exhibited a peak at 440 nm, and the long lifetime components of DPPC and PSM (7.76 and 7.77 ns, respectively) exhibited peaks at the same wavelength. This similarity arose from the competition between the collisional quenching and the hydration effects of water molecules. Herein, this phenomenon was investigated using a plot of the lifetime τ and the peak position λ (τ vs λ plot), simultaneously visualizing both effects by deconvoluting the TRES. On the basis of collisional quenching theory, the distribution of the water population per lipid (water map) was generated. According to this theory, the τ vs λ plot was applied to the water map and the calculation of the number of water molecules per lipid, which is consistent with previous reports. This approach provides novel insights for the analysis of molecular hydration states using the fluorescence of Laurdan.

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2-10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by 31 P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2-3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mode of interactions between palmitoyl lysophosphatidylcholine (palmitoyl lyso-PC) or other lysophospholipids (lyso-PLs) and palmitoyl ceramide (PCer) or other ceramide analogs in dioleoylphosphatidylcholine (DOPC) bilayers has been examined. PCer is known to segregate laterally into a ceramide-rich phase at concentrations that depend on the nature of the ceramides and the co-phospholipids. In DOPC bilayers, PCer forms a ceramide-rich phase at concentrations above 10 mol%. In the presence of 20 mol% palmitoyl lyso-PC in the DOPC bilayer, the lateral segregation of PCer was markedly facilitated (segregation at lower PCer concentrations). The thermostability of the PCer-rich phase in the presence of palmitoyl lyso-PC was also increased compared to that in the absence of palmitoyl lyso-PC. Other saturated lyso-PLs (e.g., palmitoyl lyso-phosphatidylethanolamine and lyso-sphingomyelin) also facilitated the lateral segregation of PCer in a similar manner as palmitoyl lyso-PC. When examined in the DOPC bilayer, it appeared that the association between palmitoyl lyso-PC and PCer was equimolar in nature. It is proposed that the interaction of PCer with lyso-PLs was driven by the need of ceramide to obtain a large-headgroup co-lipid, and saturated lyso-PLs were preferred co-lipids over DOPC because of the nature of their acyl chain. Structural analogs of PCer (1- or 3-deoxy-PCer) were also associated with palmitoyl lyso-PC, similarly to PCer, suggesting that the ceramide/lyso-PL interaction was not sensitive to structural alterations in the ceramide molecule. Binary complexes containing palmitoyl lyso-PC and ceramide were prepared, and these had a bilayer structure as ascertained by transmission electron microscopy. It is concluded that ceramides and lyso-PLs associated with each other both in binary bilayers and in ternary systems based on the DOPC bilayers. This association may have biological relevance under conditions in which both sphingomyelinases and phospholipase A 2 enzymes are activated, such as during inflammatory processes., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The hydration properties of the interface between lipid bilayers and bulk water are important for determining membrane characteristics. Here, the emission properties of a solvent-sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan), were evaluated in lipid bilayer systems composed of the sphingolipids D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM). The glycerophospholipids 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine were used as controls. The fluorescence properties of Laurdan in sphingolipid bilayers indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center-of-mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components based on the solvent model enabled us to identify the varieties of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Sphingolipids showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to PSM, even though DHPSM has a higher T m than PSM. Because DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups), the interfacial region of the DHPSM bilayer was expected to be in a highly polar environment. The careful analysis of Laurdan emission spectra through the four-component deconvolution in this study provides important insights for understanding the multiple polarity in the lipid membrane., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cholesterol is an essential molecule in the membranes of mammalian cells. It is known to be distributed heterogeneously within the cells, between the bilayer leaflets, as well as between lateral domains within the bilayer. However, we do not know exactly how cholesterol is distributed and what forces drive this sorting process because it extremely difficult to study using currently available methods. To further elucidate this distribution, we measured how cholesterol partitions between different phospholipid (PL) environments using different methods based on cholesterol, TopFluor-cholesterol, and cholesta-5,7,9(11)-triene-3-β-ol. Based on the obtained relative partition coefficients, we made predictions regarding how cholesterol would be distributed between lateral domains and between the inner and outer leaflets of the plasma membrane. In addition, using a trans-parinaric acid fluorescence-based method, we tested how cholesterol could influence lateral segregation through its interaction with unsaturated PLs with different headgroups. The results showed that the lower the affinity of cholesterol was for the different unsaturated PLs, the more cholesterol stimulated lateral segregation in a ternary bilayer of unsaturated PL/N-palmitoyl-D-erythro-sphingomyelin and cholesterol. Overall, the results indicate that both the distribution of cholesterol between different lipid environments and the impact of cholesterol on lateral segregation can be predicted relatively accurately from determined relative partition coefficients., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ceramides are important intermediates in sphingolipid biosynthesis (and degradation) and are normally present in only small amounts in unstressed cells. However, following the receptor-mediated activation of neutral sphingomyelinase, sphingomyelin can acutely give rise to substantial amounts of ceramides, which dramatically alter membrane properties. In this study, we have examined the role of the 1-OH and 3-OH functional groups of ceramide for its membrane properties. We have specifically examined how the oxidation of the primary alcohol to COOH or COOMe in palmitoyl ceramide (PCer) or the removal of either the primary alcohol or C(3)-OH (deoxy analogs) affected ceramides' interlipid interactions in fluid phosphatidylcholine bilayers. Measuring the time-resolved fluorescence emission of trans-parinaric acid, or its steady-state anisotropy, we have obtained information about the propensity of the ceramide analogs to form ceramide-rich domains and the thermostability of the formed domains. We observed that the oxidation of the primary alcohol to COOH shifted the ceramide's gel-phase onset concentration to slightly higher values in 1-palmitoyl-2-oleoyl- sn-3- glycero-3-phosphocholine (POPC) bilayers. Methylation of the COOH function of the ceramide did not change the segregation tendency further. The complete removal of the primary alcohol dramatically reduced the ability of 1-deoxy-PCer to form ceramide-rich ordered domains. However, the removal 3-OH (in 3-deoxy-PCer) had only small effects on the lateral segregation of the ceramide analog. The thermostability of the ceramide-rich domains in the POPC bilayers decreased in the following order: 1-OH > COOH > COOMe = 3-deoxy > 1-deoxy. We conclude that ceramide needs a hydrogen-bonding-competent functional group in the C(1) position to be able to form laterally segregated ceramide-rich domains of high packing density in POPC bilayers. The presence or absence of 3-OH was not functionally critical for ceramide's lateral segregation properties.

In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2 H NMR to elucidate the properties of nanoscopic segregated domains in stearoylsphingomyelin (SSM)/dioleoylphosphatidylcholine (DOPC) and dihydrostearoylsphingomyelin (dhSSM)/DOPC binary membranes. The results obtained from fluorescence measurements suggest the existence of gel-like domains with high fluidity in both SSM and dhSSM macroscopic gel phases. The DSC thermograms showed that DOPC destabilizes SM-rich gel-like domains to a much lesser extent compared to the same amount of cholesterol. It was also found that a stable lateral segregation occurs without cholesterol, indicating that SSM itself undergoes homophilic interactions to form small gel-like domains. 2 H NMR experiments disclosed differences in the temperature-dependent ordering of SSM/DOPC and dhSSM/DOPC bilayers; the dhSSM membrane showed less miscibility with the DOPC fluid phase, higher thermal stability, and tighter packing. In addition, the NMR results suggest the formation of mid-sized gel-like aggregates consisting of dhSSM. These differences could be accounted for by homophilic interactions, as previously reported ( Yasuda Biophys. J. 2016 , 110 , 431 - 440 ). In the absence of cholesterol, the moderately strong sphingomyelin (SM)/SM affinity results in the formation of small gel-like domains, whereas a stronger dhSSM/dhSSM affinity leads to larger gel-like domains. Considering the similar physicochemical features of SSM and dhSSM, the present results suggest that the formation of nanosized domains of SM is better characterized by homophilic interactions than by SM-cholesterol interplay. These effects are considered important to the ordered domain formation of SMs in biological membranes.

Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2 H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO - , exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.

In this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using dimonounsaturated phosphatidylcholine (PC) analogues (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUVs) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn-LUV interaction, we observed that the bilayer affinity constant (K a ) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins' N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC.

Using differential scanning calorimetry and lifetime analysis of trans-parinaric acid fluorescence, we have examined how cholesterol and cholesteryl phosphocholine (CholPC) affect gel-phase properties of palmitoyl ceramide (PCer) in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC) bilayers. By 2 H NMR, we also measured fluid-phase interactions among these lipids using deuterated analogs of POPC, PCer, and cholesterol. The PCer-rich gel phase in POPC bilayers (9:1 molar ratio of POPC to PCer) was partially and similarly dissolved (and thermostability decreased) by both cholesterol and CholPC (sterol was present equimolar to PCer, or in fourfold excess). In DOPC bilayers (4:1 DOPC/PCer molar ratio), CholPC was much more efficient in dissolving the PCer-rich gel phase when compared to cholesterol. This can be interpreted as indicating that PCer interaction with POPC was stronger than PCer interaction with DOPC. PCer-CholPC interactions were also more favored in DOPC bilayers compared to POPC bilayers. In the fluid POPC-rich phase, cholesterol increased the order of the acyl chain of d 2 -PCer much more than did CholPC. In DOPC-rich fluid bilayers, both cholesterol and CholPC increased d 2 -PCer acyl chain order, and the ordering induced by CholPC was more efficient in DOPC than in POPC bilayers. In fluid POPC bilayers, the ordering of 3-d1-cholesterol by PCer was weak. In summary, we found that in the gel phase, sterol effects on the PCer-rich gel phase were markedly influenced by the acyl chain composition of the fluid PC. The same was true for fluid-phase interactions involving the sterols. Our results further suggest that PCer did not display high affinity toward either of the sterols used. We conclude that the nature of unsaturated phospholipids (POPC versus DOPC) in bilayers has major effects on the properties of ceramide gel phases and on sterol-ceramide-phospholipid interactions in such complex bilayers., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We examined how the length of the long-chain base or the N-linked acyl chain of ceramides affected their lateral segregation in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Lateral segregation and ceramide-rich phase formation was ascertained by a lifetime analysis of trans-parinaric acid (tPA) fluorescence. The longer the length of the long-chain base (d16:1, d17:1, d18:1, d19:1, and d20:1 in N-palmitoyl ceramide), the less ceramide was needed for the onset of lateral segregation and ceramide-rich phase formation. A similar but much weaker trend was observed when sphingosine (d18:1)-based ceramide had N-linked acyl chains of increasing length (14:0 and 16:0-20:0 in one-carbon increments). The apparent lateral packing of the ceramide-rich phase, as determined from the longest-lifetime component of tPA fluorescence, also correlated strongly with the long-chain base length, but not as strongly with the N-acyl chain length. Finally, we compared two ceramide analogs with equal carbon numbers (d16:1/17:0 or d20:1/13:0) and observed that the analog with a longer sphingoid base segregated at lower bilayer concentrations to a ceramide-rich phase compared with the shorter sphingoid base analog. The gel phase formed by d20:1/13:0 ceramide also was more thermostable than the gel phase formed by d16:1/17:0 ceramide. 2 H NMR data for 10 mol % stearoyl ceramide in POPC also showed that the long-chain base was more ordered than the acyl chain at comparable chain positions and temperatures. We conclude that the long-chain base length of ceramide is more important than the acyl chain length in determining the lateral segregation of the ceramide-rich gel phase and intermolecular interactions therein., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not yet characterized isotoxin forms from the same sea anemone species.

Actinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a determinant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer-protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins' Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Saturated and unsaturated phospholipids (PLs) can segregate into lateral domains. The preference of cholesterol for saturated acyl chains over monounsaturated, and especially polyunsaturated ones, may also affect lateral segregation. Here we have studied how cholesterol influenced the lateral segregation of saturated and unsaturated PLs, for which cholesterol had a varying degree of affinity. The fluorescence lifetime of trans-parinaric acid reported the formation of ordered domains (gel or liquid-ordered (lo)) in bilayers composed of different unsaturated phosphatidylcholines, and dipalmitoyl-phosphatidylcholine or n-palmitoyl-sphingomyelin, in the presence or absence of cholesterol. We observed that cholesterol facilitated lateral segregations and the degree of facilitation correlated with the relative affinity of cholesterol for the different PLs in the bilayers. Differential scanning calorimetry and (2)H nuclear magnetic resonance showed that cholesterol increased the thermostability of both the gel and lo-domains. Increased number of double bonds in the unsaturated PL increased the order in the lo-domains, likely by enriching the ordered domains in saturated lipids and cholesterol. This supported the conclusions from the trans-parinaric acid experiments, and offers insight into how cholesterol facilitated lateral segregation. In conclusion, the relative affinity of cholesterol for different PLs appears to be an important determinant for the formation of ordered domains. Our data suggests that knowledge of the affinity of cholesterol for the different PLs in a bilayer allows prediction of the degree to which the sterol promotes lo-domain formation., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To better understand the interactions of saturated ceramides with unsaturated glycerophospholipids in bilayer membranes, we measured how palmitoyl ceramide (PCer) and dihydroceramide (dihydro-PCer, lacking the trans 4 double bond of the sphingoid base of ceramide) can interact with phosphatidylcholines (PCs) with palmitic acid in the sn-1 position and increasingly unsaturated acyl chains in the sn-2 position. The PCs were 16:0/18:1 (POPC), 16:0/18:2 (PLPC), 16:0/20:4 (PAPC), and 16:0(22:6 (PDPC). We also included di-18:1-PC (DOPC) to compare it with POPC. Because the ceramides were expected to segregate laterally to an ordered ceramide-rich phase, we determined the formation of the ordered phase using lifetime analysis of trans-parinaric acid (tPA) fluorescence. The presence of ordered domains, as indicated by tPA lifetime analysis, was verified by an analysis of tPA anisotropy as a function of temperature. The interaction between PCer and POPC was clearly more favored than interactions with DOPC, as seen from a more thermostable gel phase in POPC than in DOPC at equal ceramide content. The concentration needed for PCer gel phase formation was also lower in POPC than in the DOPC bilayers, suggesting that POPC had better miscibility in the ordered phase. The increased unsaturation of the sn-2 acyl chains of the PCs had more clear effects of dihydro-PCer segregation than on PCer segregation, and the dihydro-PCer gel phase became more thermostable as the unsaturation in the PC increased. We conclude that the interactions between ceramides and PCs were complex and affected both by the trans 4 double bond of PCer by the palmitoyl acyl in the sn-1 position and by the overall degree of unsaturation of the sn-2 acyl chain of the PCs.

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans Δ(4) double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bilayer lipids influence the lateral structure of the membranes, but the relationship between lipid properties and the lateral structure formed is not always understood. Model membrane studies on bilayers containing cholesterol and various phospholipids (PLs) suggest that high and low temperature melting PLs may segregate, especially in the presence of cholesterol. The effect of different PL headgroups on lateral structure of bilayers is also not clear. Here, we have examined the formation of lateral heterogeneity in increasingly complex (up to five-component) multilamellar bilayers. We have used time-resolved fluorescence spectroscopy with domain-selective fluorescent probes (PL-conjugated trans-parinaric acid), and (2)H NMR spectroscopy with site or perdeuterated PLs. We have measured changes in bilayer order using such domain-selective probes both as a function of temperature and composition. Our results from time-resolved fluorescence and (2)H NMR showed that in ternary bilayers, acyl chain order and thermostability in sphingomyelin-rich domains were not affected to any greater extent by the headgroup structure of the monounsaturated PLs (phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine) in the bilayer. In the complex five-component bilayers, we could not detect major differences between the different monounsaturated PLs regarding cholesterol-induced ordering. However, cholesterol clearly influenced deuterated N-palmitoyl sphingomyelin differently than the other deuterated PLs, suggesting that cholesterol favored N-palmitoyl sphingomyelin over the other PLs. Taken together, both the fluorescence spectroscopy and (2)H NMR data suggest that the complex five-component membranes displayed lateral heterogeneity, at least in the lower temperature regimen examined., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.

Sphingomyelin is an important constituent of mammalian cell membranes. Its molecular structure is N-acyl-D-erythro-sphingosylphosphorylcholine. The N-acyls in sphingomyelin often contain 16-24 carbons that are mostly saturated chains; however, the monounsaturated 24:1(Δ15c) acyl chain is also common. In addition to the more saturated nature of sphingomyelins, compared to physiologically relevant glycerophospholipids, also their hydrogen bonding properties are very different from the glycerophospholipids. Sphingomyelins form extensive intramolecular hydrogen bonds (from the 3OH of the long-chain base to phosphate oxygens of the head group), but also intermolecular hydrogen bonding involving the NH of the long-chain base are important for sphingomyelin (and sphingolipid) properties in membrane environments. Hydrogen bonding involving sphingomyelin has been shown to markedly stabilize interactions with both cholesterol and ceramide in fully hydrated bilayers. Such interactions contribute to the propensity of saturated sphingomyelin to form a liquid-ordered phase together with cholesterol, or a gel phase with saturated ceramides. The purpose of this review is to present recent experimental and computational evidence in support of the importance of hydrogen bonding for the interaction of sphingomyelin with other membrane lipids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The phospholipid acyl chain composition and order, the hydrogen bonding, and properties of the phospholipid headgroup all influence cholesterol/phospholipid interactions in hydrated bilayers. In this study, we examined the influence of hydrogen bonding on sphingomyelin (SM) colipid interactions in fluid uni- and multilamellar vesicles. We have compared the properties of oleoyl or palmitoyl SM with comparable dihydro-SMs, because the hydrogen bonding properties of SM and dihydro-SM differ. The association of cholestatrienol, a fluorescent cholesterol analog, with oleoyl sphingomyelin (OSM) was significantly stronger than its association with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in bilayers with equal acyl chain order. The association of cholestatrienol with dihydro-OSM, which lacks a trans double bond in the sphingoid base, was even stronger than the association with OSM, suggesting an important role for hydrogen bonding in stabilizing sterol/SM interactions. Furthermore, with saturated SM in the presence of 15 mol % cholesterol, cholesterol association with fluid dihydro-palmitoyl SM bilayers was stronger than seen with palmitoyl SM under similar conditions. The different hydrogen bonding properties in OSM and dihydro-OSM bilayers also influenced the segregation of palmitoyl ceramide and dipalmitoylglycerol into an ordered phase. The ordered, palmitoyl ceramide-rich phase started to form above 2 mol % in the dihydro-OSM bilayers but only above 6 mol % in the OSM bilayers. The lateral segregation of dipalmitoylglycerol was also much more pronounced in dihydro-OSM bilayers than in OSM bilayers. The results show that hydrogen bonding is important for sterol/SM and ceramide/SM interactions, as well as for the lateral segregation of a diglyceride. A possible molecular explanation for the different hydrogen bonding in SM and dihydro-SM bilayers is presented and discussed., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)