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Exosomes are small extracellular vesicles (sEVs) involved in distal cell-cell communication and cancer migration by transferring functional cargo molecules. Membrane domains similar to lipid rafts are assumed to occur in exosome membranes and are involved in interactions with target cells. However, the bilayer membrane properties of these small vesicles have not been fully investigated. Therefore, we examined the fluidity, lateral domain separation, and transbilayer asymmetry of exosome membranes using fluorescence spectroscopy. Although there were some differences between the exosomes, TMA-DPH anisotropy showing moderate lipid chain order indicated that ordered phases comprised a significant proportion of exosome membranes. Selective TEMPO quenching of the TMA-DPH fluorescence in the liquid-disordered phase indicated that 40-50% of the exosome membrane area belonged to the ordered phase based on a phase-separated model. Furthermore, NBD-PC in the outer leaflet showed longer fluorescence lifetimes than those in the inner leaflets. Therefore, the exosome membranes maintained transbilayer asymmetry with a topology similar to that of the plasma membranes. In addition, the lateral and transbilayer orders of exosome membranes obtained from different cell lines varied, probably depending on the different membrane lipid components and compositions partially derived from donor cells. As these higher membrane orders and asymmetric topologies are similar to those of cell membranes with lipid rafts, raft-like functional domains are possibly enriched on exosome membranes. These domains likely play key roles in the biological functions and cellular uptake of exosomes by facilitating selective membrane interactions with target organs.

The ginsenoside Rh2 (Rh2) is a saponin of medicinal ginseng, and it has attracted much attention for its pharmacological activities. In this study, we investigated the interaction of Rh2 with biological membranes using model membranes. We examined the effects of various lipids on the membrane-disrupting activity of Rh2 and found that cholesterol and sphingomyelin (SM) had no significant effect. Furthermore, the effects of Rh2 on acyl chain packing (DPH anisotropy) and water molecule permeability (GP

Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events that occur on cell membranes by forming glycolipid-rich membrane domains, such as lipid rafts. Lactosylceramide (LacCer) forms a highly ordered phase in model and biological membranes. However, the details of this domain remain unclear. We examined the dynamic assembly of LacCer in cholesterol-containing phase-separated membranes by fluorescence imaging and solid-state NMR. Solid-state 2H NMR of the deuterated LacCer probes in the membranes indicated that the potent LacCer–LacCer interaction significantly reduced the incorporation of cholesterol in the LacCer domains. Moreover, increasing the amount of cholesterol induced the formation of nanometer-scale LacCer domains, which can be biologically relevant. The lateral interaction of ganglioside GM3 with the epidermal growth factor receptor (EGFR) transmembrane segment was examined using fluorescence spectroscopy under membrane conditions. Lateral FRET between the NBD fluorophore on the EGFR transmembrane segment and the ATTO594 fluorophore on the GM3 head group suggested that GM3 interacts with the transmembrane segment and partially inhibits the EGFR dimerization by stabilizing the peptide monomer. More precise investigations of GSL–GSL and GSL-protein interactions occurring in membrane environments can lead to the elucidation of the cellular functions of GSL from structural points of view.

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.

S-Palmitoyl and N-myristoyl chains of Lyn cooperatively facilitate binding to the anionic lipids in membranes by extending the hydrocarbon chains deeper into the membrane interior.

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.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of fungal sterol, ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The high-resolution structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

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 Forster 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.

OSW-1, a unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane (Kapp = 0.38 μM-1) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage (r50 = 0.47) occurrence. OSW-1 showed slightly lower activity (Kapp = 0.31 μM-1; r50 = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity (Kapp = 0.017 μM-1; r50 = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 μM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 μM, r50 = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 μM, r50 = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.

Sialic acid-containing glycoconjugates are involved in important biological processes such as immune response, cancer metastasis, and viral infection. However, their chemical syntheses have been challenging, mainly due to the difficulties in the α-sialylation of oligosaccharides. Very recently, we established a completely stereoselective sialidation method using a macrobicyclic sialyl donor. Herein, we describe a rational and efficient synthesis of sialoglycolipids via direct sialylation of a glycolipid at a late-stage, based on our novel sialidation method. The synthetic method enabled the development of GM3 ganglioside analogs with various C5-modifications of the sialosyl moiety. Furthermore, the synthesized analog was subjected to solid-state 19F NMR analysis on the model membranes and it revealed the influence of cholesterol on glycan dynamics.

The core fucose, a major modification of N -glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via promotion of PD-1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. In the present study, we discovered that Dectin-1, a known anti- b -glucan lectin, recognizes the core fucose on IgGs. A combination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N -terminal asparagine residue at the glycosylation site as well as core fucose residue. Thus, Dectin-1 appears to be the first lectin-like molecule involved in the hetero-valent and specific recognition of characteristic N -glycans on antibodies.

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 Mn

Membrane proteins reside in the lipid bilayer of biomembranes and the structure and function of these proteins are closely related to their interactions with lipid molecules. Structural analyses of interactions between membrane proteins and lipids or detergents that constitute biological or artificial model membranes are important for understanding the functions and physicochemical properties of membrane proteins and biomembranes. Determination of membrane protein structures is much more difficult when compared with that of soluble proteins, but the development of various new technologies has accelerated the elucidation of the structure-function relationship of membrane proteins. This review summarizes the development of heavy atom derivative detergents and lipids that can be used for structural analysis of membrane proteins and their interactions with detergents/lipids, including their application with X-ray free-electron laser crystallography.

The NV center in a diamond is a quantum sensor with exceptional quality for highly sensitive nanoscale analysis of NMR spectra and thermometry. In this study, we investigate nanoscale phase change detection of lipid bilayers utilizing ensemble-averaged nuclear spin detection from small volume ~ (6 nm)$^{3}$, which was determined by the depth of the NV center. Analysis of nanoscale NMR signal confirm thickness of lipid bilayer to be 6.2 nm $\pm$ 3.4 nm with proton density of 65 proton/nm$^{3}$ verifying formation of lipid bilayer on top of diamond sample. Correlation spectroscopy from nanoscale volume reveals quantum oscillation at 3.06 MHz corresponding to the Larmor frequency of proton at an applied magnetic field of 71.8 mT. The result of the correlation spectroscopy was compared with the 2D molecular diffusion model constructed by Monte Carlo simulation combined with results from molecular dynamics simulation. There is a change in diffusion constant from 1.5 $\pm$ 0.25 nm$^{2}$/${\mu}$s to 3.0 $\pm$ 0.5 nm$^{2}$/${\mu}$s when the temperature changes from 26.5 $^\circ$C to 36.0 $^\circ$C. Our results demonstrate that simultaneous observation of changes in translational diffusion and temperature is possible in label-free measurements using nanoscale diamond magnetometry. Our method paves the way for label-free imaging of cell membranes for understanding its phase composition and dynamics.

Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. The inclusion of cholesterol (Chol) or ergosterol in the phosphatidylcholine membrane is known to significantly enhance the membrane affinity for theonellamide A (TNM-A). We have previously revealed that TNM-A stays in a monomeric form in dimethylsulfoxide (DMSO) solvent systems, whereas the peptide forms oligomers in aqueous media. In this study, we utilized 1H NMR chemical shift changes (Δδ1H) in aqueous DMSO solution to evaluate the TNM-A/sterol interaction. Because Chol does not dissolve well in this solvent, we used 25-hydroxycholesterol (25-HC) instead, which turned out to interact with membrane-bound TNM-A in a very similar way to that of Chol. We determined the dissociation constant, KD, by NMR titration experiments and measured the chemical shift changes of TNM-A induced by 25-HC binding in the DMSO solution. Significant changes were observed for several amino acid residues in a certain area of the molecule. The results from the solution NMR experiments, together with previous findings, suggest that the TNM-Chol complex, where the hydrophobic cavity of TNM probably incorporates Chol, becomes less polar by Chol interaction, resulting in a greater accumulation of the peptide in membrane. The deeper penetration of TNM-A into the membrane interior enhances membrane disruption. We also demonstrated that hydroxylated sterols, such as 25-HC that has higher solubility in most NMR solvents than Chol, act as a versatile substitute for sterol and could be used in 1H NMR-based studies of sterol-binding peptides.

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 Forster 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.

Cholesteryl-β-D-glucoside (ChoGlc) is a mammalian glycolipid that is expressed in brain tissue. The effects of glucosylation on the ordering and lipid interactions of cholesterol (Cho) were examined in membranes composed of N-stearoyl sphingomyelin (SSM), which is abundant in the brain, and to investigate the possible molecular mechanism involved in these interactions. Differential scanning calorimetry revealed that ChoGlc was miscible with SSM in a similar extent of Cho. Solid-state 2H NMR of deuterated SSM and fluorescent anisotropy using 1,6-diphenylhexatriene demonstrated that the glucosylation of Cho significantly reduced the effect of the sterol tetracyclic core on the ordering of SSM chains. The orientation of the sterol core was further examined by solid-state NMR analysis of deuterated and fluorinated ChoGlc analogues. ChoGlc had a smaller tilt angle between the long molecular axis (C3-C17) and the membrane normal than Cho in SSM bilayers, and the fluctuations in the tilt angle were largely unaffected by temperature-dependent mobility changes of SSM acyl chains. This orientation of the sterol core of ChoGlc leads to reduce sterol-SSM interactions. The MD simulation results suggested that the Glc moiety perturbs the SSM-sterol interactions, which reduces the umbrella effect of the phosphocholine headgroup because the hydrophilic glucose moiety resides at the same depth as an SSM amide group. These differences between ChoGlc and Cho also weaken the SSM-ChoGlc interactions. Thus, the distribution and localization of Cho and ChoGlc possibly control the stability of sphingomyelin-based domains that transiently occur at specific locations in biological membranes.

Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C′ loop of Siglec-7.

Sialic acid-containing glycoconjugates are involved in important biological processes such as immune response, cancer metastasis, and viral infection. However, their chemical syntheses have been challenging, mainly due to the difficulties in the α-sialylation of oligosaccharides. Very recently, we established a completely stereoselective sialidation method using a macrobicyclic sialyl donor. Herein, we describe a rational and efficient synthesis of sialoglycolipids via direct sialylation of a glycolipid at a late-stage, based on our novel sialidation method. The synthetic method enabled the development of GM3 ganglioside analogs with various C5-modifications of the sialosyl moiety. Furthermore, the synthesized analog was subjected to solid-state 19F NMR analysis on the model membranes and it revealed the influence of cholesterol on glycan dynamics.

N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. However, the molecular basis of GnT-V’s catalytic mechanism and substrate specificity are not fully understood. Here, we report crystal structures of human GnT-V luminal domain with a substrate analog. GnT-V luminal domain is composed of a GT-B fold and two accessary domains. Interestingly, two aromatic rings sandwich the α1-6 branch of the acceptor N-glycan and restrain the global conformation, partly explaining the fine branch specificity of GnT-V. In addition, interaction of the substrate N-glycoprotein with GnT-V likely contributes to protein-selective and site-specific glycan modification. In summary, the acceptor-GnT-V complex structure suggests a catalytic mechanism, explains the previously observed inhibition of GnT-V by branching enzyme GnT-III, and provides a basis for the rational design of drugs targeting N-glycan branching., The activity of glycosyltransferase GnT-V correlates with cancer malignancy and poor prognosis but its mechanism of action is poorly understood. Here, the authors solve crystal structures of free and substrate analog-bound GnT-V, providing insights into its catalytic mechanism and a basis for GnT-V inhibition.

Self-propagating β-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Amphidinols are polyketide metabolites produced by marine dinoflagellates and are chiefly composed of a long linear chain with polyol groups and polyolefins. Two new homologues, amphidinols 20 (AM20, 1) and 21 (AM21, 2), were isolated from Amphidinium carterae collected in Korea. Their structures were elucidated by detailed NMR analyses as amphidinol 6-type compounds with remarkably long polyol chains. Amphidinol 21 (2) has the longest linear structure among the amphidinol homologues reported so far. The congeners, particularly amphidinol 21 (2), showed weaker activity in hemolysis and antifungal assays compared to known amphidinols.

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.

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.

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 14β-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.

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.

In recent years, some examples have been reported in which gangliosides control the activity of membrane proteins, particularly RTKs. Insulin receptor is one of them and is known to interact with ganglioside GM3, resulting in the suppression of signal transduction. This interaction has attracted interest because it gives rise to type 2 diabetes by causing insulin resistance. However, there was no way to demonstrate the actual mechanism involved. Therefore, we constructed a model system that can analyze the interaction between insulin receptor and GM3 using liposome. Investigation of lipid ratio of liposome and 3D imaging made enables us to observe slight differences of peptide localization. This model would be useful for detailed analysis of electrostatic interactions.

Diosgenin (DGN), which is a sterol occurring in plants of the Dioscorea family, has attracted increasing attention for its various pharmacological activities. DGN has a structural similarity to cholesterol (Cho). In this study we investigated the effects of the common tetracyclic cores and the different side chains on the physicochemical properties of lipid bilayer membranes. Differential scanning calorimetry showed that DGN and Cho reduce the phase transition enthalpy to a similar extent. In 2H NMR, deuterated-DGN/Cho and POPC showed similar ordering in POPC bilayers, which revealed that DGN is oriented parallel to the membrane normal like Cho. It was suggested that the affinity of DGN-Cho in membrane is stronger than that of DGN-DGN or Cho-Cho interaction. 31P NMR of POPC in bilayers revealed that, unlike Cho, DGN altered the interactions of POPC headgroups at 30 mol%. These results suggest that DGN below 30 mol% has similar effects with Cho on basic biomembrane properties.

Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several 1H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2-9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the 1H NMR spectra of TNM-A with sodium dodecyl sulfate-d25 (SDS-d25) micelles and small dimyristoylphosphatidylcholine (DMPC)-d54/dihexanoylphosphatidylcholine (DHPC)-d22 bicelles in the presence of a paramagnetic quencher Mn2+. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader 1H signals of TNM-A were observed in 10mol% cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn2+ to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid-water interface (close to the C2' portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and beta 2-microglobulin (beta 2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with beta 2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 mu g/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

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 (2)H-labeled stearoyl-SM (SSM). Deuterated SSMs at the Cα, Cβ, and Cγ positions of a choline moiety were separately prepared and subjected to NMR measurements to determine the quadrupolar splitting of (2)H signals in hydrated SSM unitary and SSM/Cho (1:1) bilayers. Using (2)H NMR and (13)C-(31)P 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-Cβ, 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 Cβ and Cγ decreased more significantly, thus suggesting that Cho reduced the gauche conformation at the Cα-Cβ 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 (2)H NMR measurements showed that the chiral centers induced the slight diastereomeric excess in the SM headgroup conformation.

The chemical synthesis of the highly branched core oligosaccharides of lipooligosaccharides (LOSs) found in Campylobacter jejuni, which causes Guillain-Barre syndrome by a preceding infection, is described. The target LOS mimics, consisting of eight or nine monosaccharides, were classified into three groups as key building blocks: ganglioside-core tetra-/pentasaccharides (GM1-/GD1a-like), l-glycero-d-manno-heptose-containing trisaccharides, and 3-deoxy-d-manno-2-octulosonic acid (KDO) residues. These synthetic fragments were obtained from commercially available monosaccharides. Less obtainable l-glycero-d-manno-heptose and KDO residues, as key components of the LOSs, were synthesized from p-methoxyphenyl d-mannoside and di-O-isopropylidene-protected d-mannose, respectively. The synthesis of α-KDO glycoside, as one of the most difficult stereocontrolled glycosidic constructions, was achieved by treating a 2,3-ene derivative of KDO with phenylselenyl trifluoromethanesulfonate as a suitable α-directing reagent. All synthetic blocks were constructed through a convergent synthetic route, which resulted in the first synthesis of structurally challenging LOS core glycans containing ganglioside GM1 and GD1a-core sequences.

Amphotericin B (AmB) is a polyene macrolide antibiotic widely used to treat mycotic infections. In this paper, we focus on the role of the polyol moiety of AmB in sterol selectivity using 7-oxo-AmB, 7α-OH-AmB, and 7β-OH-AmB. The 7-OH analogs were prepared from 7-oxo-AmB. Their K + flux activity in liposomes showed that introduction of an additional ketone or hydroxy group on the polyol moiety reduces the original activity. Conformational analyses of these derivatives indicated that intramolecular hydrogen-bonding network possibly influenced the conformational rigidity of the macrolactone ring, and stabilized the active conformation in the membrane. Additionally, the flexible polyol leads to destabilization of the whole macrolactone ring conformation, resulting in a loss of sterol selectivity.

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.

Bisected N-glycans play significant roles in tumor migration and Alzheimer's disease through modulating the action and localization of their carrier proteins. Such biological functions are often discussed in terms of the conformation of the attached N-glycans with or without bisecting GlcNAc. To obtain insights into the effects of bisecting GlcNAc on glycan conformation, a systematic NMR structural analysis was performed on two pairs of synthetic N-glycans, with and without bisecting GlcNAc. The analysis reveals that terminal GlcNAcs and bisecting GlcNAc cooperate to restrict the conformations of both the α1-3 and α1-6 branches of N-glycans. 1H and 13C chemical shift comparisons suggest that bisecting GlcNAc directly modulates local conformation. Unique NOE correlations between core-mannose and the α1-3 branch mannose as well as the 3JC-H constant of the glycoside linkage indicate that bisecting GlcNAc restricts the conformation of the 1–3 branch. The angles of the glycosidic bonds between core-mannose and α1-6 branch mannose derived from 3JC-H and 3JH-H coupling constants show that terminal GlcNAcs restrict the distribution of the ψ angle to 180° and the bisecting GlcNAc increases the distribution of the ω angle +60° in the presence of terminal GlcNAcs. It is feasible that restriction of branch conformations by bisecting GlcNAc has important consequences for protein-glycan interplay and following biological events.

Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1-6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1-3 branch, while the other interacts with an N-acetylglucosamine of the α1-6 branch. In contrast, Calsepa shows 1:1 binding where α1-3 branch and core chitobiose region N-glycan interacts with lectin, while α1-6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1-3 and α1-6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

The [4+4] polymerization of an unsaturated imine, generated from the condensation of a polyamine and excess acrolein, was investigated. The polyamine was added by micropipet to acrolein, immediately yielding a mixture of the immiscible polymeric material. Microfluidic mixing was used to gradually form the soluble diazacyclooctane polymers. The polymerization reaction ultimately gave an insoluble cationic hydrogel that adhered strongly to anionic compounds on cell surfaces, including sialoglycan, and displayed a high cytotoxicity.

Background: HOP assists protein transfer in the HSP70- and HSP90-dependent protein-folding pathway. Results: HOP hydrolyzed ATP, and the region containing amino acids 1–359 (TPR1-TPR2A) was required for hydrolysis and direct interaction with ATP. Conclusion: HOP has slow ATPase activity and changes its conformation upon ATP hydrolysis. Significance: This is the first demonstration of the ATPase activity of HOP, and may enhance our understanding of the physiological function., Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

Acrolein, a toxic unsaturated aldehyde generated as a result of oxidative stress, readily reacts with a variety of nucleophilic biomolecules. Polyamines, which produced acrolein in the presence of amine oxidase, were then found to react with acrolein to produce 1,5-diazacyclooctane, a previously unrecognized but significant downstream product of oxidative stress. Although diazacyclooctane formation effectively neutralized acrolein toxicity, the diazacyclooctane hydrogel produced through a sequential diazacyclooctane polymerization reaction was highly cytotoxic. This study suggests that diazacyclooctane formation is involved in the mechanism underlying acrolein-mediated oxidative stress.