Mechanism of action of sprG1-encoded type I toxins in Staphylococcus aureus: from membrane alterations to mesosome-like structures formation and bacterial lysis.

sprG1/SprF1 is a type I toxin-antitoxin system located on Staphylococcus aureus prophage. It has previously been shown that the two toxins, SprG131 and SprG144, encoded by the sprG1 gene, are two membrane-associated peptides structured in a single α-helix. Overexpression of these two peptides leads to growth inhibition and even S. aureus death. In this study, we investigated the involvement of each peptide in this toxicity, the sequence requirements necessary for SprG131 toxicity, and the mechanism of action of these two peptides. Our findings show that both peptides, when expressed individually, are able to stop growth, with higher toxicity observed for SprG131. The combination of a hydrophobic domain and a charged domain located only at the C-terminus is necessary for this toxicity, likely to retain the orientation of the transmembrane domain. A net cationic charge for SprG131 is not essential to induce a growth defect in S. aureus. Furthermore, we established a chronology of toxic events following overexpression to gain insights into the mode of action of SprG144 and SprG131. We demonstrated that mesosome-like structures are already formed when membrane is depolarized, about 20 min after peptides induction. This membrane depolarization occurs concomitantly with a depletion of intracellular ATP, leading to S. aureus growth arrest. Moreover, we hypothesized that SprG144 and SprG131 do not form large pores in the S. aureus membrane, as ATP is not excreted into the extracellular medium, and membrane permeabilization is delayed relative to membrane depolarization. The next challenge is to identify the conditions under which SprG144 and SprG131 are naturally expressed, and to uncover their potential roles during staphylococcal growth, colonization, and infection.

Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

NMR structural elucidation of dehydrodimers resulting from oxidation of 5-O-caffeoylquinic acid in an apple juice model solution.

During apple juice and cider-making processes, phenolic compounds undergo enzymatic oxidation. 5-O-caffeoylquinic acid (CQA) is one of the major hydroxycinnamic acid derivatives and it is the preferential substrate for polyphenol oxidase (PPO) in apple juices. Consequently, CQA dehydrodimers (MW 706 Da) are among the main products resulting from CQA oxidation. CQA dehydrodimers were previously synthesized in a biomimetic apple juice model solution. Following their purification and characterization using UV-Visible spectra and mass spectrometry, the structures of seven CQA dehydrodimers were elucidated using 1H and 13C one- and two-dimensional NMR spectroscopy. Six of them exhibited dihydrobenzofuran, benzodioxane, or dihydronaphtalene skeletons, which are caffeicin-like structures. Interestingly, a new dehydrodicaffeoyldiquinic acid molecule was also characterised for which two novel structures showing a symmetric dicatechol skeleton were also proposed.

Alterins Produced by Oyster-Associated Pseudoalteromonas Are Antibacterial Cyclolipopeptides with LPS-Binding Activity.

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins' mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

Impact of polyphenols on receptor-ligand interactions by NMR: the case of neurotensin (NT)-neurotensin receptor fragment (NTS1) complex.

Ligand-receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand-receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand-receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT-NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.Communicated by Ramaswamy H. Sarma.

Novel antibiotics effective against gram-positive and -negative multi-resistant bacteria with limited resistance.

Antibiotics are a medical wonder, but an increasing frequency of resistance among most human pathogens is rendering them ineffective. If this trend continues, the consequences for public health and for the general community could be catastrophic. The current clinical pipeline, however, is very limited and is dominated by derivatives of established classes, the "me too" compounds. Here, we have exploited our recent identification of a bacterial toxin to transform it into antibiotics active on multidrug-resistant (MDR) gram-positive and -negative bacterial pathogens. We generated a new family of peptidomimetics-cyclic heptapseudopeptides-inspired from a natural bacterial peptide. Out of the 4 peptides studied, 2 are effective against methicillin-resistant Staphylococcus aureus (MRSA) in mild and severe sepsis mouse models without exhibiting toxicity on human erythrocytes and kidney cells, zebrafish embryos, and mice. These new compounds are safe at their active doses and above, without nephrotoxicity. Efficacy was also demonstrated against Pseudomonas aeruginosa and MRSA in a mouse skin infection model. Importantly, these compounds did not result in resistance after serial passages for 2 weeks and 4 or 6 days' exposure in mice. Activity of heptapseudopeptides was explained by the ability of unnatural amino acids to strengthen dynamic association with bacterial lipid bilayers and to induce membrane permeability, leading to bacterial death. Based on structure determination, we showed that cationic domains surrounded by an extended hydrophobic core could improve bactericidal activity. Because 2 peptide analogs, Pep 16 and Pep19, are effective against both MRSA and P. aeruginosa in severe sepsis and skin infection models, respectively, we believe that these peptidomimetics are promising lead candidates for drug development. We have identified potential therapeutic agents that can provide alternative treatments against antimicrobial resistance. Because the compounds are potential leads for therapeutic development, the next step is to start phase I clinical trials.

B1.12: a novel peptide interacting with the extracellular loop of the EBV oncoprotein LMP1.

Latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) plays an important role in EBV-induced cell transformation. Down-regulation of the LMP1 expression had shown promising results on cancer cell therapy. In this study, we identified by Phage display a novel peptide called B1.12 (ACPLDLRSPCG) which selectively binds to the extracellular loop (B1) of the LMP1 oncoprotein as demonstrated by molecular docking, NMR and ITC. Using an LMP1 expressing cell line, we showed that B1.12 decreased cell viability, and induced G0/G1 cell cycle arrest. In addition, the expression of A20, pAkt, and pNFkb (pRelA536) in C666-1 cells treated with B1.12 decreased compared to the untreated cells. In conclusion, we selected a novel peptide able to bind specifically to the extracellular loop of LMP1 and thus modulate its oncogenic properties.

Novel Antifungal Compounds, Spermine-Like and Short Cyclic Polylactates, Produced by Lactobacillus harbinensis K.V9.3.1Np in Yogurt.

Lactobacillus harbinensis K.V9.3.1Np was described as endowed with high antifungal activity. Most of the studies associated this activity to the produced organic acids, i.e., lactic acid, acetic acid, and hexanoic acid. The aim of this study was to purify and identify, other not yet described, antifungal molecules produced by L. harbinensis K.V9.3.1Np when used in yogurt fermentation. Active compounds were extracted through several extraction processes using organic solvents and protein precipitation. The fractions of interest were purified using flash chromatography and preparative HPLC for specific characterization. The bioactive compounds identification was performed using Nuclear Magnetic Resonance and Mass Spectrometry. Activity tests against Penicillium expansum and Yarrowia lipolytica showed that the active compounds from L. harbinensis K.V9.3.1Np are benzoic acid and a polyamine identified as a spermine analog, which has not been reported earlier. However, the highest activity was shown by a mixture of short (n = 2-5) polycyclic lactates. Our overall results demonstrate the efficiency of the proposed extraction/purification approach. The new compounds described here have promising antifungal activities but further studies are still needed to decipher their mode of action and production pathways. Even though, they present an interesting potential application in food, feed, as well as, in pharmaceutical industries and could serve as alternative to chemical additives.

Human Dystrophin Structural Changes upon Binding to Anionic Membrane Lipids.

Scaffolding proteins play important roles in supporting the plasma membrane (sarcolemma) of muscle cells. Among them, dystrophin strengthens the sarcolemma through protein-lipid interactions, and its absence due to gene mutations leads to the severe Duchenne muscular dystrophy. Most of the dystrophin protein consists of a central domain made of 24 spectrin-like coiled-coil repeats (R). Using small angle neutron scattering (SANS) and the contrast variation technique, we specifically probed the structure of the three first consecutive repeats 1-3 (R1-3), a part of dystrophin known to physiologically interact with membrane lipids. R1-3 free in solution was compared to its structure adopted in the presence of phospholipid-based bicelles. SANS data for the protein/lipid complexes were obtained with contrast-matched bicelles under various phospholipid compositions to probe the role of electrostatic interactions. When bound to anionic bicelles, large modifications of the protein three-dimensional structure were detected, as revealed by a significant increase of the protein gyration radius from 42 ± 1 to 60 ± 4 Å. R1-3/anionic bicelle complexes were further analyzed by coarse-grained molecular dynamics simulations. From these studies, we report an all-atom model of R1-3 that highlights the opening of the R1 coiled-coil repeat when bound to the membrane lipids. This model is totally in agreement with SANS and click chemistry/mass spectrometry data. We conclude that the sarcolemma membrane anchoring that occurs during the contraction/elongation process of muscles could be ensured by this coiled-coil opening. Therefore, understanding these structural changes may help in the design of rationalized shortened dystrophins for gene therapy. Finally, our strategy opens up new possibilities for structure determination of peripheral and integral membrane proteins not compatible with different high-resolution structural methods.

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions.

Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin family proteins. However, the effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain, as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exons 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for DMD.

Selectivity Modulation and Structure of α/aza-ß3 Cyclic Antimicrobial Peptides.

Potent and selective antimicrobial cyclic pseudopeptides (ACPPs) mixing α- and aza-ß3 -amino acids were developed. Cyclopseudopeptide sequences were designed to investigate the impact of some intrinsic molecular parameters on their biological activities. Fine changes in the nature of the side chains strongly modulated the selectivity of the ACPPs with regard to hemolysis versus antimicrobial activity. The conformational preference of such compounds in various media was extensively studied, and the typical structure of cyclic α/aza-ß3 -pseudopeptides is described for the first time. Interestingly, such scaffolds are stabilized by successive inverse γ- and N-N turns (hydrazino turns), a unique feature due to the aza-ß3 residues. The α-amino acid side chains form a cluster on one face of the ring, while the aza-ß3 -amino acid side chains are projected around the ring in the equatorial orientation. Such structural data are particularly valuable to fine-tune the bioactivity of these ACPPs by a structure-based approach.

Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis.

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

Contrast-Matched Isotropic Bicelles: A Versatile Tool to Specifically Probe the Solution Structure of Peripheral Membrane Proteins Using SANS.

Obtaining structural information on integral or peripheral membrane proteins is currently arduous due to the difficulty of their solubilization, purification, and crystallization (for X-ray crystallography (XRC) application). To overcome this challenge, bicelles are known to be a versatile tool for high-resolution structure determination, especially when using solution and/or solid state nuclear magnetic resonance (NMR) and, to a lesser extent, XRC. For proteins not compatible with these high-resolution methods, small-angle X-ray and neutron scattering (SAXS and SANS, respectively) are powerful alternatives to obtain structural information directly in solution. In particular, the SANS-based approach is a unique technique to obtain low-resolution structures of proteins in interactions with partners by contrast-matching the signal coming from the latter. In the present study, isotropic bicelles are used as a membrane mimic model for SANS-based structural studies of bound peripheral membrane proteins. We emphasize that the SANS signal coming from the deuterated isotropic bicelles can be contrast-matched in 100% D2O-based buffer, allowing us to separately and specifically focus on the signal coming from the protein in interaction with membrane lipids. We applied this method to the DYS-R11-15 protein, a fragment of the central domain of human dystrophin known to interact with lipids, and we were able to recover the signal from the protein alone. This approach gives rise to new perspectives to determine the solution structure of peripheral membrane proteins interacting with lipid membranes and might be extended to integral membrane proteins.

Bioactive Metabolites from the Deep Subseafloor Fungus Oidiodendron griseum UBOCC-A-114129.

Four bioactive compounds have been isolated from the fungus Oidiodendron griseum UBOCC-A-114129 cultivated from deep subsurface sediment. They were structurally characterized using a combination of LC-MS/MS and NMR analyses as fuscin and its derivatives (dihydrofuscin, dihydrosecofuscin, and secofuscin) and identified as polyketides. Albeit those compounds were already obtained from terrestrial fungi, this is the first report of their production by an Oidiodendron species and by the deepest subseafloor isolate ever studied for biological activities. We report a weak antibacterial activity of dihydrosecofuscin and secofuscin mainly directed against Gram-positive bacteria (Minimum Inhibitory Concentration (MIC) equal to Minimum Bactericidal Concentration (MBC), in the range of 100 µg/mL). The activity on various protein kinases was also analyzed and revealed a significant inhibition of CDC2-like kinase-1 (CLK1) by dihysecofuscin.

Minor Pyranonaphthoquinones from the Apothecia of the Lichen Ophioparma ventosa.

Four new quinonoid naphthopyranones, ophioparmin (1), 4-methoxyhaemoventosins (2a and 2b), and 4-hydroxyhaemoventosin (3), together with anhydrofusarubin lactone (4) and haemoventosin (5) were isolated from the fruiting bodies of Ophioparma ventosa, a crustose lichen. Their structures were determined by spectroscopic analyses, and the absolute configurations of 1 and 2 were elucidated through experimental and calculated electronic circular dichroism analyses. Compounds 1, 2, and 5 exhibited moderate to strong antioxidant activities. The main pigment haemoventosin exhibited significant cytotoxicity toward a panel of nine cell lines.

Two distinct conformational states of Mycobacterium tuberculosis virulent factor early secreted antigenic target 6 kDa are behind the discrepancy around its biological functions.

Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.

Heterobi- and -trimetallic Ion Pairs of Zirconocene-Based Isoselective Olefin Polymerization Catalysts with AlMe3.

The reactivity towards AlMe3 of discrete cationic ansa-zirconocenes 2 a,b that are ubiquitously used in isoselective propylene polymerization and based on [{Ph(H)C(3,6-tBu2-Flu)(3-tBu-5-Et-Cp)}ZrMe2)] {Cp-Flu} and rac-[{Me2Si-(2-Me-4-Ph-Ind)2}ZrMe2] {SBI} was scrutinized. The first example of a structurally characterized Group 4 metallocene AlMe3 adduct (3 b) is reported. In the presence of excess AlMe3, the {SBI}-based AlMe3 adduct 3 b undergoes a slow decomposition via C-H activation in a bridging methyl unit to yield a new species (4 b) with a trimetallic {Zr(µ-CH2)(µ-Me)AlMe(µ-Me)AlMe2} core. EXSY NMR data for the process 2 b⇄3 b→4 b suggest very rapid and reversible binding of an additional AlMe3 molecule onto AlMe3 adduct 3 b. The resulting heterotrimetallic species intermediates exchange of methyl groups between different metal centers and slowly undergoes the C-H activation reaction towards 4 b.

Easy and quantitative access to Fe(II) and Fe(III) di(aryl)alkynylphosphine oxides featuring [Fe(dppe)Cp*] endgroups: terminal P=O functionality blocks the dimerisation of the Fe(III) derivatives.

A series of paramagnetic di(aryl)alkynylphosphine oxides [PF6] featuring an open-shell [Fe(κ(2)-dppe)(η(5)-C5Me5)](+) endgroup were obtained by oxidation of their neutral Fe(II) parents 3a-c, themselves obtained in a simple and nearly quantitative fashion from the corresponding Fe(II) metallophosphines 1a-c. The new organometallic radicals were characterised by NMR and ESR and were shown to be perfectly stable in solution, in contrast to species such as 1a-b[PF6] which readily dimerise.

Peptidic ligands to control the three-dimensional self-assembly of quantum rods in aqueous media.

The use of peptidic ligands is validated as a generic chemical platform allowing one to finely control the organization in solid phase of semiconductor nanorods originally dispersed in an aqueous media. An original method to generate, on a macroscopic scale and with the desired geometry, three-dimensional supracrystals composed of quantum rods is introduced. In a first step, nanorods are transferred in an aqueous phase thanks to the substitution of the original capping layer by peptidic ligands. Infrared and nuclear magnetic resonance spectroscopy data prove that the exchange is complete; fluorescence spectroscopy demonstrates that the emitter optical properties are not significantly altered; electrophoresis and dynamic light scattering experiments assess the good colloidal stability of the resulting aqueous suspension. In a second step, water evaporation in a microstructured environment yields superstructures with a chosen geometry and in which nanorods obey a smectic B arrangement, as shown by electron microscopy. Incidentally, bulk drying in a capillary tube generates a similar local order, as evidenced by small angle X-ray scattering.

Sterols associated with small unilamellar vesicles (SUVs): intrinsic mobility role for 1H NMR detection.

Small unilamellar vesicles (SUVs) of phospholipids are often used as a membrane model system for studying the interaction of molecules. When using NMR under the standard liquid-state conditions, SUV phospholipid proton spectra can be recorded, exhibiting sharp signals. This is not only because of the fast vesicular tumbling but also because of the combination of this tumbling with the individual motion of the lipids inside the bilayer. This appears evident because addition of cholesterol is responsible of broader resonances because of the slowing down of the lipid motion. On the other hand, no (1)H signal is detected for cholesterol in the bilayer. This lack of detection of the inserted molecules explains why generally SUVs are not considered as a good model for NMR studies under the standard liquid-state conditions. Here, we use two other sterols in order to demonstrate that an increase of the molecular mobility inside the bilayer could allow the detection of their proton resonances. For desmosterol and lanosterol, which show higher mobility inside the bilayer, with increasing lateral diffusion rates, (1)H sterol signals are detected in contrast to cholesterol. For the fast diffusing lanosterol, no significant improvement in detection is observed using deuterated lipids, demonstrating that homonuclear dipolar coupling is fully averaged out. Furthermore, in the case of low mobility such as for cholesterol, the use of a fast magic angle spinning probe is shown to be efficient to recover the full proton spectrum.