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.

LnDOTA-d8 , a versatile chemical-shift thermometer for 2 H solid-state NMR.

2 H solid-state nuclear magnetic resonance (NMR) is a method for examining the mobility and orientation of molecules in the field of biophysics. In studies on lipid bilayer membranes, 2 H NMR is often adopted to detect a phase transition from the gel to the liquid-crystal phase, which is observed as a change in spectral shape, and to evaluate the ordering of lipid alkyl chains using quadrupole coupling values. Because the mobility of membrane lipids is highly temperature dependent, precise temperature control is a prerequisite for evaluating the physical properties of membranes. Generally, NMR instruments monitor the temperature of the variable temperature (VT) gas. The temperature inside the sample tube and the VT gas match only when the heat generated by the radio frequency (rf) pulse emitted from the coil or magic angle spinning is significantly lower than the cooling capacity of the VT gas. In other words, the sample temperature inside the tube depends on the measurement method. Therefore, in this study, we took advantage of temperature-dependent changes in the chemical shift of a paramagnetic metal-ligand complex. We designed and synthesized a deuterated ligand complex and evaluated its temperature dependence as a thermometer for 2 H solid-state NMR spectroscopy. We chose Tb, Dy, Ho, and Er as the paramagnetic central metals. We then measured the 2 H NMR spectrum of each metal complex and confirmed the 2 H chemical shift to be temperature dependent. Furthermore, with the use of the thermometer molecule with Er, we succeeded in accurately evaluating the segmental melting of an alkyl chain in lipid bilayers with 0.1°C accuracy.

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.

Conformation and Orientation of Branched Acyl Chains Responsible for the Physical Stability of Diphytanoylphosphatidylcholine.

Diphytanoylphosphatidylcholine (DPhPC) is a synthetic phospholipid in which two methyl-branched acyl chains are introduced into the glycerol moiety, mimicking phospholipids of eukaryotic and eubacterial origins. The lipid bilayers of DPhPC reproduce the outstanding physical properties of methyl-branched lipids that occur in archaeal membranes. DPhPC is commonly used as the base lipid in biophysical experiments, particularly for recording ion-channel currents. However, the dynamics of lipid molecules that induces their useful physical properties is still unclear. In this study, we examined the conformation and orientation of the methyl-branched acyl chain of DPhPC in a membrane using 2H nuclear magnetic resonance (NMR) measurements of the synthetic lipid with a high stereochemical purity and molecular dynamics (MD) simulations. Deuterium-labeled 3',3'-CD3,D-DPhPC (2) and 7',7'-CD3,D-DPhPC (3) showed the characteristic quadrupole splitting width in the 2H NMR spectra, which corresponded to the bent orientation reported for the archaeal lipid PGP-Me [Yamagami, M., et al. (2019) Biochemistry 58, 3869-3879]. However, MD simulations, which reproduced the 2H NMR results well, unveiled the unknown features of DPhPC in the membrane; DPhPC has a chain-specific average orientation, where two bent orientations with upward and downward methyl groups occur at positions C3 and C7 of the sn-1 and sn-2 chains of DPhPC, respectively. These MD and NMR results reveal that these two bent orientations define the average orientation of DPhPC for the shallow part of the acyl chains, which is considered to be an important factor in the stability of DPhPC membranes.

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.

Cholesterol-Induced Conformational Change in the Sphingomyelin Headgroup.

Sphingomyelin (SM) and cholesterol (Cho) are the important lipids for the formation of biologically functional membrane domains, lipid rafts. However, the interaction between Cho and the headgroup of SM remains unclear. In this study, we performed solid-state NMR experiments to reveal the Cho effects on the headgroup conformation using 2H-labeled stearoyl-SM (SSM). Deuterated SSMs at the Cα, Cß, and Cγ positions of a choline moiety were separately prepared and subjected to NMR measurements to determine the quadrupolar splitting of 2H signals in hydrated SSM unitary and SSM/Cho (1:1) bilayers. Using 2H NMR and 13C-31P REDOR data, the conformation and orientation of the choline moiety were deduced and compared with those derived from molecular dynamics simulations. In SSM unitary bilayers, three torsional angles in the phosphocholine moiety, P-O-Cα-Cß, were found to be consecutive +gauche(g)/+g/+g or -g/-g/-g. The orientation and conformation of the SSM headgroup were consistent with the results of our molecular dynamics simulations and the previous results on phosphatidylcholines. The quadrupolar coupling at the α methylene group slightly increased in the presence of Cho, and those at the Cß and Cγ decreased more significantly, thus suggesting that Cho reduced the gauche conformation at the Cα-Cß torsion. The conformational ensemble in the presence of Cho may enhance the so-called umbrella effect of the SSM headgroup, resulting in the stabilization of Cho near the SM molecules by concealing the hydrophobic Cho core from interfacial water. We also examined the effect of the chiral centers at the sphingosine chain to the headgroup conformation by determining the enantiomeric excess between the diastereomeric +g/+g/+g and -g/-g/-g conformers using (S)-Cα-deuterated and (R)-Cα-deuterated SSMs. Their 2H NMR measurements showed that the chiral centers induced the slight diastereomeric excess in the SM headgroup conformation.

Average Conformation of Branched Chain Lipid PGP-Me That Accounts for the Thermal Stability and High-Salinity Resistance of Archaeal Membranes.

The average conformation of the methyl-branched chains of archaeal lipid phosphatidyl glycerophosphate methyl ester (PGP-Me) was examined in a hydrated bilayer membrane based on the 2H nuclear magnetic resonance (NMR) of enantioselectively 2H-labeled compounds that were totally synthesized for the first time in this study. The NMR results in combination with molecular dynamics simulations revealed that the PGP-Me chain appeared to exhibit behavior different from that of typical membrane lipids such as dimyristoylphosphatidylcholine (DMPC). The C-C bonds of the PGP-Me chain adopt alternative parallel and tilted orientations to the membrane normal as opposed to a DMPC chain where all of the C-C bonds tilt in the same way on average. This characteristic orientation causes the intertwining of PGP-Me chains, which plays an important role in the excellent thermal and high-salinity stabilities of archaeal lipid bilayers and membrane proteins.

The Amphotericin B-Ergosterol Complex Spans a Lipid Bilayer as a Single-Length Assembly.

Amphotericin B (AmB) is a polyene macrolide antibiotic clinically used as an antifungal drug. Its preferential complexation with ergosterol (Erg), the major sterol of fungal membranes, leads to the formation of a barrel-stave-like ion channel across a lipid bilayer. To gain a better understanding of the mechanism of action, the mode of lipid bilayer spanning provides essential information. However, because of the lack of methodologies to observe it directly, it has not been revealed for the Erg-containing channel assembly for many years. In this study, we disclosed that the AmB-Erg complex spans a lipid bilayer with a single-molecule length, using solid-state nuclear magnetic resonance (NMR) experiments. Paramagnetic relaxation enhancement by Mn2+ residing near the surface of lipid bilayers induced the depth-dependent decay of 13C NMR signals for individual carbon atoms of AmB. We found that both terminal segments, the 41-COOH group and C38-C40 methyl groups, come close to the lipid bilayer surfaces, suggesting that the AmB-Erg complex spans a palmitoyloleoylphosphatidylcholine (POPC) bilayer with a single-molecule length. Molecular dynamics simulation experiments further confirmed the stabilization of the AmB-Erg complex as a single-length spanning complex. These results provide experimental evidence of the single-length complex incorporated in the membrane by making thinner a POPC-Erg bilayer that mimics fungal membranes.

The Perpendicular Orientation of Amphotericin B Methyl Ester in Hydrated Lipid Bilayers Supports the Barrel-Stave Model.

The clinically important antibiotic amphotericin B (AmB) is a membrane-active natural product that targets membrane sterol. The antimicrobial activity of AmB is generally attributed to its membrane permeabilization, which occurs when a pore is formed across a lipid bilayer. In this study, the molecular orientation of AmB was investigated using solid-state nuclear magnetic resonance (NMR) to better understand the mechanism of antifungal activity. The methyl ester of AmB (AME) labeled with NMR isotopes, d3-AME, and its fluorinated and/or 13C-labeled derivatives were prepared. All of the AmB derivatives showed similar membrane-disrupting activities and ultraviolet spectra in phospholipid liposomes, suggesting that their molecular assemblies in membranes closely mimic those of AmB. Solid-state 2H NMR measurements of d3-AME in a hydrated membrane showed that the mobility of AME molecules depends on concentration and temperature. At a 1:5:45 AME:Erg:dimyristoylphosphatidylcholine ratio, AME became sufficiently mobilized to observe the motional averaging of quadrupole coupling. On the basis of the rotational averaging effect of 19F chemical shift anisotropy, 2H quadrupolar splitting, and 13C-19F dipolar coupling of 14ß-F-AMEs, we deduced that the molecular axis of AME is predominantly parallel to the normal of a lipid bilayer. This result supports the barrel-stave model as a molecular assembly of AmB in membranes.

Side-chain deuterated cholesterol as a molecular probe to determine membrane order and cholesterol partitioning.

Cholesterol is an essential and ubiquitous component in mammalian cell membranes. However, its distributions and interactions with phospholipids are often elusive, partly because chemical modifications for preparing cholesterol probes often cause significant perturbations in its membrane behavior. To overcome these problems, a 2H-labeled probe (24-d-cholesterol), which perfectly retained the original membrane properties, was synthesized by a stereoselective introduction of 2H into the side chain of cholesterol. A deuterium label at the side-chain more sensitively reflects membrane fluidity than the conventional labeling at the 3 position of a sterol core (3-d-cholesterol), thus providing 24-d-cholesterol with desirable properties to report membrane ordering. Solid state 2H NMR of 24-d-cholesterol with sphingomyelins (SM) and unsaturated phosphatidylcholine in the bilayer membranes clearly revealed the partitioning ratio of cholesterol in the raft-like liquid ordered (Lo) phase and the liquid disordered phase based on cholesterol interactions with surrounding lipids in each phase. This probe turned out to be superior to the widely used 3-d-cholesterol; e.g., 24-d-cholesterol clearly revealed a 10 mol% difference in the Lo distribution ratios of cholesterol between palmitoyl-SM and stearoyl-SM. The comprehensive use of 24-d-cholesterol in solid state 2H NMR will disclose the cholesterol-lipid interactions, distribution ratio of cholesterol, and membrane ordering in model bilayers as well as more complicated biological membranes.

Small structural alterations greatly influence the membrane affinity of lipophilic ligands: Membrane interactions of bafilomycin A1 and its desmethyl derivative bearing 19F-labeling.

Molecular behavior under bilayer membrane environments is one of the important research topics concerning how organic molecules exert their biological activities when interacting with cellular membranes. However, chemistry-based approaches to this property have not been successful when compared with the structural biological strategy on ligand-receptor interactions. Here, we investigated the molecular behavior of the lipophilic ATPase inhibitor bafilomycin A1 and its derivatives under a lipid environment from a chemical point of view. Our results revealed significant differences in membrane affinity and dynamics among ligands having different inhibitory potencies, suggesting the specific contribution of ligand-membrane interactions to their biological activity.

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.

Synthesis and Complete Structure Determination of a Sperm-Activating and -Attracting Factor Isolated from the Ascidian Ascidia sydneiensis.

For the complete structure elucidation of an endogenous sperm-activating and -attracting factor isolated from eggs of the ascidian Ascidia sydneiensis ( Assydn-SAAF), its two possible diastereomers with respect to C-25 were synthesized. Starting from ergosterol, the characteristic steroid backbone was constructed by using an intramolecular pinacol coupling reaction and stereoselective reduction of a hydroxy ketone as key steps, and the side chain was introduced by Julia-Kocienski olefination. Comparison of the NMR data of the two diastereomers with those of the natural product led to the elucidation of the absolute configuration as 25 S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.

Stable C-N axial chirality in 1-aryluracil scaffold and differences in in vitro metabolic clearance between atropisomers of PDE4 inhibitor.

We report herein the stable C-N axial chirality in a 1-phenyl-6-aminouracil scaffold owing to the presence of various functional groups at the ortho-position of the N(1)-phenyl group. Racemic 1-phenyl-6-aminouracils were first separated by chiral HPLC or converting them to the corresponding diastereomers using a chiral resolving agent. We then determined the rotational barrier of each atropisomer by a thermal racemization method and found that these compounds have rotational barriers similar to other C-N axially chiral biaryls. In addition, there was a good correlation between the rotational barriers and van der Waals radii of an ortho-substituent of the N(1)-phenyl group. To explore the possibility of the chiral 1-phenyl-6-aminouracil scaffold as a drug lead, we synthesized both atropisomers as phosphodiesterase-4 inhibitors 10. The atropisomers showed significantly different metabolic stabilities while their PDE4 inhibitory activities were somewhat similar. This finding demonstrates the potential utility of stable C-N bond atropisomers in the development of chiral drugs.

The Structure of the Bimolecular Complex between Amphotericin B and Ergosterol in Membranes Is Stabilized by Face-to-Face van der Waals Interaction with Their Rigid Cyclic Cores.

Amphotericin B (AmB) is a polyene macrolide antibiotic isolated from Streptomyces nodosus. The antifungal activity of AmB can be attributed to the formation of an ion-channel assembly in the presence of ergosterol (Erg), in which there are two different AmB-Erg orientations, parallel and antiparallel, as reported previously. In this study, to elucidate the structures of those AmB-Erg complexes based on solid-state nuclear magnetic resonance, a (19)F-labeled AmB derivative was newly prepared by a hybrid synthesis that utilized degradation products from the drug. Using the 2-(trimethylsilyl)ethoxymethyl (SEM) group as the protecting group for the carboxylic acid moiety of AmB, the fully deprotected labeled AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments in hydrated lipid bilayers. The results indicated the coexistence of parallel and antiparallel orientations for AmB and Erg pairing, at a ratio of 7:3. A total of six distances between AmB and Erg were successfully obtained. Geometry analysis using the distance constraints derived from the REDOR experiments provided the plausible AmB-Erg complex structure for both the parallel and antiparallel interactions. The flat macrolide of AmB and the tetracyclic core of Erg closely contacted in a face-to-face manner, thus maximizing the van der Waals interaction between the two molecules. This interaction can be attributed to the coexistence of both the parallel and antiparallel orientations.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain.

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes.

Deuterium NMR of raft model membranes reveals domain-specific order profiles and compositional distribution.

In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (Lo) and liquid-disordered (Ld) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting Lo and Ld domains. We also found that the quadrupole profile of the Lo phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that (2)H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the Lo and Ld domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our (2)H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.

Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol's affinity to amphotericin B in membrane.

The interaction of amphotericin B (AmB) with fungal ergosterol (Erg) is stronger than its interaction with mammalian cholesterol (Cho), and this property of AmB as an antifungal drug is thought to be responsible for its selective toxicity toward fungi. However, the mechanism by which AmB recognizes the structural differences between sterols, particularly minor difference in the sterol alicyclic portion, is largely unknown. Thus, to investigate the mode of interaction between AmB and the sterol core, we assessed the affinity of AmB to various sterols with different alicyclic structures. Ion flux assays and UV spectral measurements clearly revealed the importance of the Δ7-double bond of the sterol B-ring for interaction with the drug. AmB showed lower affinity for triene sterols, which have double bonds at the Δ5, Δ7, and Δ9 positions. Intermolecular distance measurements by (13)C{(19)F} rotational echo double resonance (REDOR) revealed that the AmB macrolide ring is in closer contact with the steroid core of Erg than it is with the Cho core in the membrane. Conformational analysis suggested that an axial hydrogen atom at C7 of Δ5-sterol (2, 6) and the protruded A-ring of Δ5,7,9-sterol (4, 8) sterically hampered face-to-face contact between the van der Waals surface of the sterol core and the macrolide of AmB. These results further suggest that the α-face of sterol alicycle interacts with the flat macrolide structure of AmB.

Formation of Gel-like Nanodomains in Cholesterol-Containing Sphingomyelin or Phosphatidylcholine Binary Membrane As Examined by Fluorescence Lifetimes and (2)H NMR Spectra.

In this study, we measured the time-resolved fluorescence of trans-parinaric acid (tPA), steady-state fluorescence anisotropy of diphenylhexatriene (DPH), and (2)H NMR of 10,10-d2-stearoyl lipids in stearoyl sphingomyelin with cholesterol (SSM/Chol) and l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine with Chol (PSPC/Chol) binary membranes. The results suggest that the membrane order obtained from the fluorescence experiments shows a similar temperature dependency as those of the (2)H NMR data. More importantly, the time-resolved fluorescence data implied the presence of at least two types of domains, cholesterol-poor gel-like domains (CPGLD) and cholesterol-enriched liquid-ordered (Lo) domains. These domains appear on a nano-to-micro second time scale for both SSM-Chol and PSPC-Chol membranes. The relative size of the gel-like domain was also estimated from the temperature-dependent lifetime measurements and (2)H NMR spectral changes. The results imply that the size of the gel-like domains is very small, probably on the nanometer scale, and smaller in SSM-Chol membrane than those in PSPC-Chol bilayers, which could account for the higher thermal stability of SM-Chol membranes. The present study demonstrates that gel-like nanodomains occur in SM-Chol binary membrane even with Chol content of over 33 mol %, which has been thought to consist exclusively of Lo phase, implying that not only Lo domains but also gel-like nanodomains are important for formation of lipid-ordered phase in SM-Chol and PC-Chol membranes.