Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers.

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 2H NMR spectra of 10',10'-d2-LacCer and 18',18',18'-d3-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.

Depth-Dependent Segmental Melting of the Sphingomyelin Alkyl Chain in Lipid Bilayers.

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.

Oligomerization of Sticholysins from Förster Resonance Energy Transfer.

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.

Preparation of Nitrogen Analogues of Ceramide and Studies of Their Aggregation in Sphingomyelin Bilayers.

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: CerN3, CerNH2, and CerNHAc, in which the 1-OH group of ceramide is substituted with a nitrogen functionality. CerNH2 and CerNHAc are capable of forming hydrogen bonds in their headgroups, whereas CerN3 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 CerN3 was similar to that of normal ceramide, while the CerNH2-rich phase showed a slightly looser chain packing due to the formation of CerNH3+. Although the domain formation of CerN3 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.

Sphingomyelin Acyl Chains Influence the Formation of Sphingomyelin- and Cholesterol-Enriched Domains.

The segregation of lipids into lateral membrane domains has been extensively studied. It is well established that the structural differences between phospholipids play an important role in lateral membrane organization. When a high enough cholesterol concentration is present in the bilayer, liquid-ordered (Lo) domains, which are enriched in cholesterol and saturated phospholipids such as sphingomyelin (SM), may form. We have recently shown that such a formation of domains can be facilitated by the affinity differences of cholesterol for the saturated and unsaturated phospholipids present in the bilayer. In mammalian membranes, the saturated phospholipids are usually SMs with different acyl chains, the abundance of which vary with cell type. In this study, we investigated how the acyl chain structure of SMs affects the formation of SM- and cholesterol-enriched domains. From the analysis of trans-parinaric acid fluorescence emission lifetimes, we could determine that cholesterol facilitated lateral segregation most with the SMs that had 16 carbon-long acyl chains. Using differential scanning calorimetry and Förster resonance energy transfer techniques, we observed that the SM- and cholesterol-enriched domains with 16 carbon-long SMs were most thermally stabilized by cholesterol. The Förster resonance energy transfer technique also suggested that the same SMs also form the largest Lo domains. In agreement with our previously published data, the extent of influence that cholesterol had on the propensity of lateral segregation and the properties of Lo domains correlated with the relative affinity of cholesterol for the phospholipids present in the bilayers. Therefore, the specific SM species present in the membranes, together with unsaturated phospholipids and cholesterol, can be used by the cell to fine-tune the lateral structure of the membranes.

Sphingomyelins and ent-Sphingomyelins Form Homophilic Nano-Subdomains within Liquid Ordered Domains.

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.

Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus.

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 ß-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.

Functional and Structural Variation among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus.

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.

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers.

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 31P 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.

Solvatochromic Modeling of Laurdan for Multiple Polarity Analysis of Dihydrosphingomyelin Bilayer.

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 Tm 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.

The Affinity of Sterols for Different Phospholipid Classes and Its Impact on Lateral Segregation.

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-ß-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.

Lateral Segregation of Palmitoyl Ceramide-1-Phosphate in Simple and Complex Bilayers.

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.

Impact of Acyl Chain Mismatch on the Formation and Properties of Sphingomyelin-Cholesterol Domains.

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 (Tm)) phospholipid, a saturated (high-Tm) 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-Tm 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-Tm and high-Tm phospholipids and the effects of low- and high-Tm 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.

Natural Ceramides and Lysophospholipids Cosegregate in Fluid Phosphatidylcholine Bilayers.

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 A2 enzymes are activated, such as during inflammatory processes.

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction.

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.

Membrane Localization and Lipid Interactions of Common Lipid-Conjugated Fluorescence Probes.

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 2H and 31P NMR spectroscopy in the solid state. In addition, using 2D 1H 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. 1H 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.

Lipid-Surrounding Water Molecules Probed by Time-Resolved Emission Spectra of Laurdan.

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.

Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation.

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 2H 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.

Nanosized Phase Segregation of Sphingomyelin and Dihydrosphigomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol.

In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2H 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. 2H 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.

Preparation and Membrane Properties of Oxidized Ceramide Derivatives.

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.