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Bicelles are modelmembranes generally made of long-chain dimyristoylphosphatidylcholine(DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensivelyused in the study of membrane interactions and structure determinationof membrane-associated peptides, since their composition and morphologymimic the widespread PC-rich natural eukaryotic membranes. At lowDMPC/DHPC (q) molar ratios, fast-tumbling bicellesare formed in which the DMPC bilayer is stabilized by DHPC moleculesin the high-curvature rim region. Experimental constraints imposedby techniques such as circular dichroism, dynamic light scattering,or microscopy may require the use of bicelles at high dilutions. Studieshave shown that such conditions induce the formation of small aggregatesand alter the lipid-to-detergent ratio of the bicelle assemblies.The objectives of this work were to determine the exact compositionof those DMPC/DHPC isotropic bicelles and study the lipid miscibility.This was done using 31P nuclear magnetic resonance (NMR)and exploring a wide range of lipid concentrations (2–400 mM)and qratios (0.15–2). Our data demonstratehow dilution modifies the actual DMPC/DHPC molar ratio in the bicelles.Care must be taken for samples with a total lipid concentration ≤250mM and especially at q∼ 1.5–2, sincemoderate dilutions could lead to the formation of large and slow-tumblinglipid structures that could hinder the use of solution NMR methods,circular dichroism or dynamic light scattering studies. Our results,supported by infrared spectroscopy and molecular dynamics simulations,also show that phospholipids in bicelles are largely segregated onlywhen q> 1. Boundaries are presented within whichcontrol of the bicelles’ qratio is possible.This work, thus, intends to guide the choice of qratio and total phospholipid concentration when using isotropicbicelles. [ABSTRACT FROM AUTHOR]

Abstract: Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate 2H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. 2H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences. [Copyright &y& Elsevier]

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo 2H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(−) bacteria membranes. We also investigated the effect of marennine using 31P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis. [Display omitted] • The action mechanism of marennine on Vibrio splendidus is studied in vivo by 2H solid-state NMR. • The specific interaction with phospholipids is studied by 31P solid-state NMR with model membranes. • Marennine stiffens phospholipid acyl chains, even at low concentration. • The lipid ordering effect is greater for bacteria sampled in the stationary phase. • Marennine does not disrupt bacterial membranes but would act via alteration of the membrane fluidity. [ABSTRACT FROM AUTHOR]

The compound "marennine" is a blue–green pigment produced by the benthic microalgae Haslea ostrearia, with pathogenicity reduction activities against some bacteria and promising potential as a natural pigment in seafood industries. After decades of research, the chemical family of this compound still remains unclear, mainly because structural studies were impaired by the presence of co-extracted compounds in marennine isolates. To improve the purity of marennine extract, we developed a novel extraction method using a graphitic stationary phase, which provides various advantages over the previous procedure using tandem ultrafiltration. Our method is faster, more versatile, provides a better crude yield (66%, compared to 57% for ultrafiltration) and is amenable to upscaling with continuous photobioreactor cultivation. Our goal was to take advantage of the modulable surface properties of the graphitic matrix by optimizing its interactions with marennine. As such, the effects of organic modifiers, pH and reducing agents were studied. With this improvement on marennine purification, we achieved altogether the isolation of a fucoidan-related, sulfated polysaccharide from blue water. Characterization of the polysaccharides fraction suggests that roughly half of UV-absorbing compounds could be isolated from the marennine crude extracts. The identification of sulfated polysaccharides could be a major breakthrough for marennine purification, providing targeted isolation techniques. Likewise, the added value of Haslea ostrearia and the role of polysaccharides in previous marennine chemical characterization and bioactivity studies remain to be determined. [ABSTRACT FROM AUTHOR]

We present a new membrane mimetic system using a membrane softening detergent commonly known as Tween 80 (TW80), to form oriented systems for solid-state NMR applications. TW80 is a fatty acid ester (oleate) of sorbitan polyethoxylate and a mild non-ionic surfactant. Phosphatidylcholine (PC)/TW80 model membrane systems were characterized by solid-state NMR and FTIR spectroscopy. 31P and 2H NMR spectra showed that DMPC (14:0) and DPPC (16:0) self-assemble with TW80 to form oriented structures, and maintain alignment over a wide range of molar ratios and temperatures. The addition of lanthanide ions revealed that the membrane alignment can be flipped from parallel to perpendicular with respect to the magnetic field direction. Using 15N solid-state NMR and a labeled model transmembrane peptide, we showed that TW80-based membranes can be employed to determine the peptide orientation in the magnetic field, which is useful for structural determination. Altogether, our work showed that TW80 could be exploited for direct and efficient membrane protein extraction and to enhance membrane and membrane protein orientation without using a detergent removal step. This approach could be extended to a wide range of membranes including native ones. Unlabelled Image • Phosphatidylcholine model membranes with improved magnetic orientation can be formed by adding the mild surfactant Tween-80 • The magnetic orientation is sufficient to yield highly resolved 31P and 2H solid-state NMR spectra • A transmembrane peptide incorporated in such membranes is shown to be well oriented • The membrane systems, with and without proteins, can be flipped to align the bilayer normal with the magnetic field using paramagnetic ions [ABSTRACT FROM AUTHOR]

• Mussel tissues accumulated a mean of 26 µg STX eq. 100 g–1 after a 120 h exposure to cultured A. catenella. • Regardless of saxitoxin concentration or immune cell type, most cells remained alive and non-apoptotic. • The neutral lipid content of mussel immune cells decreased by a factor of two with A. catenella exposure. • A. catenella exposure affected the energy metabolism and osmolyte composition of mussel immune cells. The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes – the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella. [ABSTRACT FROM AUTHOR]

Protein misfolding and amyloid deposition are associated with numerous diseases. The detailed characterization of the proteospecies mediating cell death remains elusive owing to the (supra)structural polymorphism and transient nature of the assemblies populating the amyloid pathway. Here we describe the identification of toxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). While N21Q filaments share structural properties with cytocompatible fibrils, including the 4.7 Å inter-strand distance and β-sheet-rich conformation, they concurrently display characteristics of oligomers, such as low thioflavin-T binding, high surface hydrophobicity and recognition by the A11 antibody, leading to high potency to disrupt membranes and cause cellular dysfunction. The toxic oligomer-like conformation of N21Q fibrils, which is preserved upon elongation, is transmissible to naïve IAPP. These stable fibrils expanding the conformational diversity of amyloid assemblies represent an opportunity to elucidate the structural basis of amyloid disorders. Nguyen et al identified cytotoxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). They presented evidence to show that these stable fibrils expand the conformational diversity of amyloid assemblies, which represents an opportunity to elucidate the structural basis of amyloid disorders. [ABSTRACT FROM AUTHOR]

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and β-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells. [Display omitted] • IAPP induces the clustering of lipid vesicles derived from mammalian cell plasma membranes. • Membrane clusters exhibit colocalization between lipid bilayer vesicles. • Perturbation of cell-derived vesicles induced by IAPP can be detected by FRET. • FRET signal obtained from damaged cell-derived lipid vesicles can be kinetically followed using a microplate format. • A new assay is reported to investigate cell plasma membrane perturbation under biologically relevant conditions. [ABSTRACT FROM AUTHOR]

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membranebound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr- 545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels. [ABSTRACT FROM AUTHOR]

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase. [Display omitted] • A fast and facile lipid profile characterization pipeline is proposed. • Complete bacterial lipid profiles are determined at different growth stages. • Membrane fluidity is measured for bacteria under various labeling conditions. • Optimized lipid labeling protocol is established for whole-cell solid-state NMR. • Bacteria should be harvested at the early stationary phase. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) are incorporated in many consumer products and have a high-releasing potential in aquatic environments. In these environments, AgNPs could interact with colloidal natural organic matter (NOM) affecting their behavior and fate. Aggregation kinetics of AgNPs were conducted at 10 NaCl concentrations during 2 h with 2.5 or 25 mgNOM L−1. Four (4) colloidal NOM fractions were extracted from 50 m3 of St.-Lawrence estuarine waters: fulvic acids (SLFA), humic acids (SLHA), hydrophilic (SL4ILE), and hydrophobic (SL4OBE) fractions. The addition of NOM to AgNP dispersion in nanopure water induced a small increase in AgNP size depending on NOM concentration and fraction used. The presence of both salt and NOM significantly altered the size of silver aggregates. With added 2.5 mgNOM L−1, more than 90% of particles have a size smaller than 200 nm, independently of the NOM fraction used. At 25 mgNOM L−1, the particle size distribution was strongly affected by the nature of NOM fractions. Our results show that NOM with highly aromatic carbon content such as SLFA and SLHA promoted the formation of larger aggregates with a hydrodynamic diameter larger than 200 nm. A high content in non-polar aliphatic carbon as observed in SL4ILE and SL4OBE stabilized aggregates at a size below 200 nm. This study highlights the importance of the chemical composition of NOM fractions and their content in non-polar aliphatic and aromatic carbons as they are involved in hydrophobic effects and strongly influence the aggregate size distribution. Sulfur, also present at low concentration NOM fractions, appears to be involved in the aggregation of AgNPs, but its action mechanism was not determined. [Display omitted] • Effects of concentration of natural organic matter on the size of aggregates. • At 2.5 mgNOM L−1, more than 90% of particles have a size smaller than 200 nm. • At 25 mgNOM L−1, the particle size distribution was affected by the nature of NOM. • NOM with high content of aromatic carbon promote aggregates > 200 nm. • NOM with high content of non-polar aliphatic carbon promote aggregates < 200 nm. [ABSTRACT FROM AUTHOR]

Fluorine-19 is an ideal nucleus for studying biological systems using nuclear magnetic resonance (NMR) due to its rarity in biological environments and its favorable magnetic properties. In this work, we used a mixture of monofluorinated palmitic acids (PAs) as tracers to investigate the molecular interaction of the fluorinated drug rosuvastatin in model lipid membranes. More specifically, PAs labeled at the 4 th and 8 th carbon positions of their acyl chains were co-incorporated in phospholipid bilayers to probe different depths of the hydrophobic core. First, the 19 F chemical shift anisotropy (CSA) indicative of membrane fluidity, was simultaneously determined for fatty acids (FAs) and the fluorinated drug using either slow magic-angle spinning (MAS) 1D 19 F solid-state NMR (SS-NMR) or MAS 2D 19 F- 19 F SS-NMR with CSA recoupling. Membrane heterogeneity and selective partitioning of rosuvastatin into fluid regions could thus be evidenced. We then examined the possibility of mapping intermolecular distances in bilayers, in both the fluid and gel phases, using 19 F- 19 F and 1 H- 19 F correlation experiments by SS-NMR using MAS. Spatial correlations were evidenced between the two PAs in the gel phase, while contacts between the statin and the lipids were detected in the fluid phase. This work paves the way to mapping membrane-active molecules in intact membranes, and stresses the need for new labeling strategies for this purpose., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed 19 F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the 19 F chemical shift anisotropy, monitored through a CF bond order parameter (S CF ), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning 19 F ssNMR spectra with and without 1 H decoupling, by studying alterations in the second spectral moment (M 2 ) as well as the 19 F isotropic chemical shift, linewidth, T 1 , and T 2 relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T 1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using 19 F ssNMR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers., (© 2024. The Author(s).)

Nature is rich with examples of highly specialized biological materials produced by organisms for functions, including defense, hunting, and protection. Along these lines, velvet worms (Onychophora) expel a protein-based slime used for hunting and defense that upon shearing and dehydration forms fibers as stiff as thermoplastics. These fibers can dissolve back into their precursor proteins in water, after which they can be drawn into new fibers, providing biological inspiration to design recyclable materials. Elevated phosphorus content in velvet worm slime was previously observed and putatively ascribed to protein phosphorylation. Here, we show instead that phosphorus is primarily present as phosphonate moieties in the slime of distantly related velvet worm species. Using high-resolution nuclear magnetic resonance (NMR), natural abundance dynamic nuclear polarization (DNP), and mass spectrometry (MS), we demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with glycans linked to large slime proteins, while transcriptomic analyses confirm the expression of 2-AEP synthesizing enzymes in slime glands. The evolutionary conservation of this rare protein modification suggests an essential functional role of phosphonates in velvet worm slime and should stimulate further study of the function of this unusual chemical modification in nature.

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H + buffering, regulation of Ca 2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1 -/- ) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca 2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1 -/- resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1 -/- resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca 2+ peaks and slowed Ca 2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca 2+ handling and excitation-contraction coupling., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo 2 H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2 H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A variety of lipids that differ by their chains and headgroups are found in biomembranes. In addition to studying the overall membrane phase, determination of the structure, dynamics, and headgroup conformation of individual lipids in the mixture would be of great interest. We have thus developed, to our knowledge, a new approach using solid-state 31 P NMR, magic-angle spinning, and chemical-shift anisotropy (CSA) recoupling, using an altered version of the recoupling of chemical shift anisotropy (ROCSA) pulse sequence, here penned PROCSA. The resulting two-dimensional spectra allowed the simultaneous measurement of the isotropic chemical shift and CSA of each lipid headgroup, thus providing a valuable measure of its dynamics and structure. PROCSA was applied to mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in various relative proportions, to mimic bacterial membranes and assess the respective roles of lipids in shaping these bilayers. The results were interpreted in terms of membrane topology, lipid propensity to adopt various phases or conformations, and lipid-lipid miscibility. Our results showed that PG dictates the lipid behavior when present in a proportion of 20 mol % or more. A small proportion of PG is thus able to impose a bilayer structure to the hexagonal phase forming PE. We discuss the requirement for lipids, such as PE, to be able to adopt non-bilayer phases in a membrane., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The molecular structure of the blue mussel Mytilus edulis whole anchoring threads was studied by two-dimensional (13)C solid-state NMR on fully labeled fibers. This unique material proves to be well ordered at a molecular level despite its heterogeneous composition as evidenced by the narrow measured linewidths below 1.5 ppm. The spectra are dominated by residues in collagen environments, as determined from chemical shift analysis, and a complete two-dimensional assignment (including minor amino acids) was possible. The best agreement between predicted and experimental backbone chemical shifts was obtained for collagen helices with torsion angles (-75°, +150°). The abundant glycine and alanine residues can be resolved in up to five different structural environments. Alanine peaks could be assigned to collagen triple-helices, β-sheets (parallel and antiparallel), β-turns, and unordered structures. The use of ATR-FTIR microscopy confirmed the presence of these structural environments and enabled their location in the core of the thread (collagen helices and antiparallel β-sheets) or its cuticle (unordered structures). The approach should enable characterization at the molecular level of a wide range of byssus macroscopic properties.

As part of ongoing activities toward the design of potent and selective ligands against galactoside-binding proteins from animal, bacterial, and plant lectins, a systematic investigation involving the synthesis and binding evaluations of a series of original β-C-galactopyranoside mimetics is described. The multivalent presentation of partly optimized candidates on various dendritic scaffolds through Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAc) has also been achieved. Biophysical investigations based on isothermal titration calorimetry (ITC) have indicated a dissociation constant in the low micromolar range for the best optimized monovalent conjugate (K(d)=37 μM). The results thus confirmed that stable C-galactosides could represent efficient synthetic glycomimetics of natural α-linked oligosaccharidic inhibitors of PA-IL lectin (Lec A) from the pathogenic Pseudomonas aeruginosa. Striking enhancements in the avidity of the glycoconjugates were also observed for tri-, hexa-, and nonavalent derivatives, among which the most potent exhibited dissociation constants below 500 nM, corresponding to a 400-fold increase in affinity compared with the β-D-Gal-O-Me used as reference. To deepen our understanding of the binding mode of the best glycomimetics involved in the recognition process, molecular modeling studies, docking calculations, and NMR diffusion measurements have been performed. Although favorable complementary interactions induced by the addition of the hydrophobic aglycon might explain the affinity enhancement, experimental determination of the size and the topology of the multivalent conjugates further supported the formation of aggregative complexes as a major multivalent binding mode. This work represents a systematic and comprehensive study towards a thorough understanding of the protein-carbohydrate interactions involved in Pseudomonas aeruginosa infection, and as such should prove useful for the development of stable and optimized anti-adhesive agents., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)