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BACKGROUND: Hypertriglyceridemia is the third most common cause of acute pancreatitis (AP). It has been shown that hypertriglyceridemia aggravates the severity and related complications of AP; however, detailed analyses of large cohorts are contradictory. Our aim was to investigate the dose-dependent effect of hypertriglyceridemia on AP. METHODS: AP patients over 18 years old who underwent triglyceride measurement within the initial three days were included into our cohort analysis from a prospective international, multicenter AP registry operated by the Hungarian Pancreatic Study Group. Data on 716 AP cases were analyzed. Six groups were created based on the highest triglyceride level (

Elucidation and improvement of the blood coagulant properties of heparin are the focus of intense research. In this study, we performed conformational analysis using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations on the heparin pentasaccharide analogue idraparinux, its disulfonatomethyl analogue, which features a slightly improved blood coagulation property, and a trisulfonatomethyl analogue, in which the activity has been totally abolished. As the ring conformation of the G subunit has been suggested as a major determinant of the biological properties, we analyzed the sugar ring conformations and dynamics of the interglycosidic linkages. We found that the conformation of the G ring is dominated by the 2SO skewed boat next to the 1C4 chair in all three derivatives. Both the thermodynamics and the kinetics of the conformational states were found to be highly similar in the three derivatives. Molecular kinetic analysis showed that the 2SO skewed boat state of the G ring is equally favorable in the three analogues, resulting in similar 2SO populations. Also, the transition kinetics from the 1C4 chair to the 2SO skewed boat was found to be comparable in the derivatives, which indicates a similar energy barrier between the two states of the G subunit. We also identified a slower conformational transition between the dominant 4C1 chair and the boat conformations on the E subunit. Both G and E ring flips are also accompanied by changes along the interglycosidic linkages, which take place highly synchronously with the ring flips. These findings indicate that conformational plasticity of the G ring and the dominance of the 2SO skewed boat populations do not necessarily warrant the biological activity of the derivatives and hence the impact of other factors also needs to be considered.

International audience; We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Model experiments under laboratory conditions were carried out to assess the accumulation tendency of Zr from a silica-zirconia core–shell nanoparticles, synthesised for the assay. Acute exposition tests were conducted with Daphnia magna and Danio rerio and the accumulation tendency of the nano and the molecular form was compared. Significant elevation of Zr was found in the tissue of the test organisms treated by the NP compared to the control, however, the retention of zebrafish was lower than that of the daphnids. Increased level of bioconcentration factor and the strong correlation of the redundancy analysis data suggest accumulation tendency in D. magna, yet long-term experiments are required to prove and further assess the environmental risk of food-chain bioconcentration. The BCF results were under the REACH limit for the D. rerio groups indicating the low short-term accumulation tendency of Zr from the NP. However, Zr level was significantly higher in zebrafish individuals originating from the SiO2@ZrO2 NP exposed treatments compared to the groups supplemented by the same concentration of ZrOCl2, which did not result in the elevation of Zr in fish tissue. The trace element homeostasis of D. rerio was not affected by the acute SiO2@ZrO2 NP exposition and no lethality occurred.

With the aim to develop compounds able to target multiple metabolic pathways and, thus, to lower the chances of drug resistance, we investigated the anti-trypanosomal activity and selectivity of a series of symmetric diglycosyl diselenides and disulfides. Of 18 compounds tested the fully acetylated forms of di-β-D-glucopyranosyl and di-β-D-galactopyranosyl diselenides (13 and 15, respectively) displayed strong growth inhibition against the bloodstream stage of African trypanosomes (EC50 0.54 μM for 13 and 1.49 μM for 15) although with rather low selectivity (SI, Graphical abstract Image 1, Highlights • Acetylated diglycosyl diselenides inhibit the proliferation of infective Trypanosoma brucei. • A galactosyl derivative impairs parasite' glucose consumption and redox homeostasis. • Diglycosyl diselenides react covalently with glutathione under mild conditions.. • Acetylated diglycosyl diselenides represent multitarget antitrypanosomal candidates.

International audience; We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro–(S)-Pro) segment as a “templating” unit, frequently used in the design of β-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis–trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF 3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced β-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro–(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans–cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro–(R)-TfmOxa–(S)-Val-OtBu β-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans–trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro–(R)-TfmOxa template fulfilled all crucial β-turn-inducer criteria.

Silica@zirconia core@shell oxide nanoparticles of approx. 50 nm diameter were developed in view of an application as a novel carrier with adjuvant activities for cancer vaccines. To this end, the aim was to cover the surface of the nanocarrier by potent single stranded DNA (ssDNA) type immune stimulators used in cancer vaccination. Prior to characterizing the binding of ssDNA to the nanocarrier surface, the adsorption of deoxynucleoside monophosphate building blocks (dNMPs) was studied. After optimization of conditions for synthesis, solvent exchange and surface modification, the effects and possible interference of several buffers, such as HEPES, PIPES, MOPS and MOPSO, were also investigated. Formation of a new compound was revealed by UV–VIS spectroscopy during the recording of zeta potential vs. pH titration curves in the presence of HEPES and PIPES buffer in the neutral-acidic pH region. We identified this new species as gold ions etched from the electrode of the zeta cuvette, stabilized by the buffers via chelation, which is then followed by their conversion into gold nanoparticles.

A biologically active peptide toxin containing four diselenide bonds was synthesized. The diselenide network and its dynamics were disclosed using a combined NMR and MD approach., Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin containing four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve potential therapeutic use of the toxin, we have designed a double substituted analog, [N17A/F32T]-AnTx, which showed comparable Kv1.3 affinity to the wild-type peptide, but also a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chemical synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace, by chemical synthesis, all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly, the key pharmacological properties of the Sec-[N17A/F32T]-AnTx are retained since the peptide is functionally active. We also propose here a combined experimental and theoretical approach including NOE- and 77Se-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-[N17A/F32T]-AnTx. Using this combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed characterization of the conformational dynamics around each disulfide/diselenide bridge.

Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces.

International audience; G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β2 -adrenergic receptor (β2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β2 AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of β2 AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a β-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production.

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted into the α-MSH pharmacophore, His(6)-Phe(7)-Arg(8)-Trp(9)-domain. Replacement of His(6) by an aminoindoloazepinone (Aia) or aminobenzazepinone (Aba) moiety led to hMC4R and hMC5R selective agonist and antagonist ligands, respectively (tetrapeptides 1 to 3 and 4, respectively). In peptides 1 to 3 and depending on the para-substituent of the d-Phe residue in position 2, the activity goes from allosteric partial agonism (1, R = H) to allosteric full agonism (2, R = F) and finally allosteric partial agonism (3, R = Br).

The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKa(H) are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6 degrees C and is key in modulating the pKa(H) of the imidazolium group. The general features, prerequisites and generic character of the new pKa(H)-regulating motif are described.

We report an efficient and adaptable method for the microencapsulation of active ingredients by a polydimethylsiloxane (PDMS) shell material. The core–shell microcapsules were obtained by phase separation between the core component and the PDMS shell components after repartitioning of the common solvent THF between the PDMS/core material phase and the water phase. For the shell components, two commercially available functional PDMS polymers containing thiol and vinyl side groups were used. Photo-cross-linking in the presence of 2,2-dimethoxy-2-phenylacetophenone (DMPA) by thiol–ene radical addition was used to form a PDMS-thioether cross-linked shell. Variation of the PDMS component thiol to ene ratio resulted in different functionalities on the microcapsules surface and in the bulk, which was analyzed by attenuated total-reflection infrared spectroscopy (ATR-IR) and high-resolution magic-angle NMR-spectroscopy (HR-MAS NMR). Organically modified silica particles were mixed into the PDMS shell, resulting i...

The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins.

PubMed ID: 29058690, Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein., VE 196/1-1, HE 7456/1-1, Jo 1324/1-1 U.S. Public Health Service: DK-56338 National Institutes of Health: DK076683 National Institute of Diabetes and Digestive and Kidney Diseases: DK-98120 National Research Foundation of Korea, NRF: 2015R1D1A1A01056685 Department of Science and Technology, Government of Kerala NAS LPDS-2015-07 Fudan University, We are grateful to the families and study participants for their contributions. We thank the Yale Center for Mendelian Genomics (U54HG006504) for WES analysis. FH is a William E. Harmon Professor of Pediatrics. This research was supported by the NIH (DK076683, to FH); the Young Scholars Program of Children’s Hospital of Fudan University (to JR); Basic Science Research Program through the National Research Foundation of Korea 2015R1D1A1A01056685 (to HYG); DFG fellowships (VE 196/1-1, to ATvdV; Jo 1324/1-1, to TJS; and HE 7456/1-1, to TH); the German National Academy of Sciences Leopoldina (LPDS-2015-07, to EW); the Egyptian Group for Orphan Renal Diseases (EGORD) (to NAS); the Department of Science and Technology, Government of India (DST-SERB, to MAJ); the National Institute of Diabetes and Digestive and Kidney Diseases (DK-98120, to SK); and the Public Health Service (DK-56338, to SK).

The endoplasmic reticulum malectin is a highly conserved protein in the animal kingdom that has no counterpart so far in lower organisms. We recently determined the structure of its conserved domain and found a highly selective binding to Glc(2)Man(9)GlcNAc(2), an intermediate of N-glycosylation. In our quest for putative ligands during the initial characterization of the protein, we noticed that the malectin domain is highly specific for diglucosides but quite tolerant towards the linkage of the glucosidic bond. To understand the molecular requirements for the observed promiscuity of the malectin domain, here we analyze the binding to a range of diglucosides through comparison of the protein chemical shift perturbation patterns and the saturation transfer difference spectra of the ligands including two maltose-mimicking drugs. A comparison of the maltose-bound structure of the malectin domain with the complex of the native ligand nigerose reveals why malectin is able to tolerate such a diversity of ligands.

N-Glycosylation starts in the endoplasmic reticulum (ER) where a 14-sugar glycan composed of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc3Man9GlcNAc2) is transferred to nascent proteins. The glucoses are sequentially trimmed by ER-resident glucosidases. The Glc3Man9GlcNAc2moiety is the substrate for oligosaccharyltransferase; the Glc1Man9GlcNAc2and Man9GlcNAc2intermediates are signals for glycoprotein folding and quality control in the calnexin/calreticulin cycle. Here, we report a novel membrane-anchored ER protein that is highly conserved in animals and that recognizes the Glc2-N-glycan. Structure determination by nuclear magnetic resonance showed that its luminal part is a carbohydrate binding domain that recognizes glucose oligomers. Carbohydrate microarray analyses revealed a uniquely selective binding to a Glc2-N-glycan probe. The localization, structure, and binding specificity of this protein, which we have named malectin, open the way to studies of its role in the genesis, processing and secretion of N-glycosylated proteins.

When it comes to failing or injured tissues and organs, patients often end up on waiting lists for tissue or even organ transplantation negatively affecting the patient’s quality of life. The multidisciplinary research field of tissue engineering may offer more innovative ways to replace or ideally regenerate failing tissues and organs. A widely used material in this research field is gelatin because of its biocompatibility and interesting hydrogel forming properties. However, at body temperature gelatin’s mechanical properties are greatly reduced due to the dissolution of collagen-like triple helices. With the aim to obtain materials that retain their mechanical properties at body temperature, we propose to combine sodium alginate and methacrylamide-modified gelatin (Gel-MOD) in the form of a graft copolymer to obtain a material that closely resembles the extracellular matrix. The obtained materials can be cross-linked via three distinct pathways including cation mediated, temperature mediated or via covalent bond formation after UV irradiation in the presence of a photo-initiator. The current contribution covers the synthesis of the above mentioned alginate-graft-gelatin copolymers and the characterization of the resulting hydrogels. The materials developed are highly hydrophilic, showing high gel fractions and satisfactory mechanical properties. Moreover the attained storage moduli were tunable by divalent cation addition (72–275% increase at 21 °C, 42–405% increase at 40 °C). One formulation was found to outperform Gel-MOD in terms of mechanical properties at 40 °C, thus indicating the proposed strategy can be used to improve the mechanical properties of gelatin-based hydrogels. Moreover, in vitro biocompatibility assays indicated that cell adhesion and proliferation improves with increasing gelatin content. The present paper illustrates that the developed triple cross-linkable materials are suitable cell carriers, promising to be applied for biomedical purposes.

Here, we present a modified CPMG-HSQMBC experiment which is capable to reduce the detrimental phase twists in the “long-range” connectivity multiplets caused by proton–proton couplings. We demonstrate that concerted CPMG pulse trains applied on both nuclei in the starting CPMG-INEPT transfer step can considerably be improved by composite π pulses that compensate for pulse imperfections and off-resonance effects. Experimental optimization of the interpulse delay within the CPMG cycle was found to be crucial in order to achieve the best possible “decoupling” of homonuclear coupling modulation.

Peptidic lipopolysaccharide (LPS) antagonists are the subject of intensive research. We report an NMR and modeling study of LALF-14 (GCKPTFRRLKWKYKCG), a synthetic cyclized fragment of the limulus anti-LPS factor (LALF) comprising residues 36-47. In a mixture with LPS we observed the transferred NOE effect and derived the LPS-bound structure of LALF-14. Neither the free nor the LPS-bound peptide displays NOEs indicative of a beta-sheet-like structure that is adopted by the fragment in the full-size protein. However, docking calculations show that the former structure is not a prerequisite for binding of LALF-14 to LPS.

One-bond heteronuclear and two-bond homonuclear residual dipolar couplings measured at methylene or amine sites can be utilized as long-range constraints in structure determination of molecules as well as to facilitate characterization of local conformation by stereospecific assignment of diastereotopic protons. We present two J-modulated HMQC type experiments to measure the one-bond heteronuclear dipolar coupling contributions of geminal protons individually. In addition two-bond homonuclear residual dipolar couplings between the diastereotopic protons are also obtained.

The voltage-gated Kv1.3 K+ channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.

The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both l- and d-amino acids. Remarkably, the Leu5 amino acid-centrally located in the nonapeptide sequence-is the sole residue found to possess either an l or d configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR-restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self-assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L- or D-Leu5.

Self-consistent, full assignments of the 1 H and 13 C NMR spectra are reported for the macrocyclic antibiotics oligomycin-B and -C. Solution conformations, deduced from proton–proton and proton–carbon-13 coupling constants as well as from chemical shift considerations, are compared with X-ray data. While coupling data are compatible with a fair degree of similarity between the overall conformations in the solid and solution states, small but definite differences are apparent in the “northern” part of the macrocycle (C-6 to C-12) of oligomycin-B. On the other hand, conformational differences, relative to the same region, have been detected between oligomycin-B and oligomycin-C in solution; this is likely due to steric crowding between bulky substiuents at C-9 and C-12 in oligomycin-B.

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(β- d -galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11 μM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.

The conformations of several β1→β1′ diglycosyl disulfides were investigated by NMR and computational methods. Experimental data, such as NOEs, proton–proton and proton–carbon-13 coupling constants, measured for solutions in DMSO, are in good agreement with values obtained by MD simulations in explicit DMSO. The disulfide torsion angles (C1–S–S–C1′) preferentially sample values close to either +90° or −90° (+g or −g) and appear as the main metric that determines the conformational behavior of these glycomimetics. There is more conformational freedom around the C1–S and C1′–S′ bonds (Φ and Ω torsions, respectively) and population cluster analysis allowed to identify up to four allowed conformational regions for each of the +g or −g forms. Population analysis of the hydroxylic group rotamers, based on proton–proton and proton–carbon-13 couplings as well as on calculated hydrogen bonding statistics, did not reveal any significant intramolecular hydrogen bonds in DMSO solution.

The family of cysteine rich proteins (CRP) comprises three closely homologous members that have been reported to interact with α-actinin. Muscular LIM protein (MLP/CRP3), the skeletal muscle variant, was originally discovered as a positive regulator of myogenesis and is suggested to be part of the stretch sensor of the myofibril through its interaction with telethonin (T-Cap). We determined the structure of both LIM domains of human MLP by nuclear magnetic resonance spectroscopy. We confirm by 15N relaxation measurements that both LIM domains act as independent units and that the adjacent linker regions are fully flexible. With the published structures of CRP1 and CRP2, the complete family has now been structurally characterized.

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys47 and Cys95 and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4Å resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys47, Gln82, and Trp137 do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys107 against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys97 and Lys99 play a crucial role in the interaction with tryparedoxin.

6 páginas, 5 figuras, 1 esquema -- PAGS nros. 17148-17153, Competition binding experiments are used in NMR-based screenings to match up to the binding site with that of a known ligand and to determine the strength of the interaction, thus providing a ranking of hits according to receptor affinity. These competition titration experiments must use a reference ligand for which the binding site on the receptor and the affinity of the interaction is known. These experiments rely on the observation of separate signals of the reference and hit compounds, which is very often hampered by signal overlap. Here, we present a combination of isotope editing and filtering schemes with saturation transfer difference (STD) experiments that allows the separation of the STD signals of the labeled reference ligand from that of the natural abundance hit compound even in the case of severe signal overlap. Thus, the measurement of well-defined titration curves in competition STD titration experiments is feasible and allows the quantitative determination of binding constants. Note that the method requires the availability of the reference ligand in an NMR-active, stable-isotope-labeled form, We thank Thomas Schallus for the expression vector of malectin and for sharing ideas prior to publication. K.E.K and G.B. acknowledge the support of the Hungarian Scientific Research Fund, OTKA NK-68578. C.M.-G. acknowledges the DFG Grant Mu1606/2-2. We also thank the scientific exchange program between CSIC (Spain) and Hungary for travel support

The effect of the magnetic field strength on the magnitude of residual dipolar couplings introduced by dilute liquid crystal media was investigated. One-bond heteronuclear residual dipolar couplings and residual deuterium quadrupolar splitting of the water were monitored at various static magnetic fields. It is suggested that the value of residual anisotropic NMR parameters resulting from exchange between ordered and non-ordered molecules decreases with increasing magnetic fields.

We report a G-BIRD(r) modified coupled HSQC experiment for the accurate determination of one-bond heteronuclear residual dipolar couplings. The G-BIRD(r) module has been employed to refocus the long-range coupling evolution of the heteronucleus during the t1 frequency labeling period. As a result, the crosspeaks obtained are split by only the direct one-bond coupling that can be extracted by measuring simple frequency differences between singlet maxima. Additionally the decoupling of long-range multiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence resulting in a significant sensitivity and resolution improvement.

Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties. Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized. We have obtained full assignments of the 1 H and 13 C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions. NOESY/ROESY experiments have provided interproton distance restraints that were used in distance geometry modelling calculations to derive conformational preferences for each of these molecules. These data were supplemented with information available from chemical shifts, temperature dependence of amide proton shifts and proton–proton scalar couplings. Analysis of the results suggest that the lipophilic substituents attached to the Dap 3 -e amino group of the parent PGM molecule introduce changes to the conformational preferences of the peptide moiety. In PGM electrostatic interactions between charged end groups apparently promote folded conformations with participation of the long Dap side chain. Derivatives wherein such interactions are suppressed by acylation of the Dap 3 -e amino group are characterized by more extended conformations of the peptide chain. The new synthetic derivatives exhibit biological properties similar to those of the parent PGM. This may indicate that peripheral parts of the peptide chain such as the C-terminal and end groups of the long Dap side chain do not significantly contribute to the binding to receptors or enzymes participating in the biochemical interactions referred to above.

Sulfonomethyl mimics of 2-ulosonic acids were prepared by addition of the ethyl methanesulfonate carbanion on aldonolactone derivatives, and these were converted into new analogues of the sialyl Lewis X tetrasaccharide.

A general scheme is proposed for eliminating disturbing signals of some protecting groups from the 2D NMR spectra of oligosaccharides by making use of a band-selective suppression–restoration sequence that serves as a preparation step to various 2D NMR experiments.

Erratum Erratum to ‘‘Conformational preferences in diglycosyl disulfides: NMR and molecular modeling studies’’ [Carbohydr. Res. 346 (2011) 2612–2621] Krisztina Feher a,b, , Richard P. Matthews c,d, , Katalin E. Kover , Kevin J. Naidoo c,d,⇑, Laszlo Szilagyi a,⇑ Department of Organic Chemistry, University of Debrecen, Pf. 20, H-4010 Debrecen, Hungary Department of Inorganic Chemistry, University of Debrecen, Pf. 21, H-4010 Debrecen, Hungary c Scientific Computing Research Unit, University of Cape Town, Rondebosch 7701, South Africa Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa

Quantum dot core–shell structures are essential to superior optical properties. However, Cd-based quantum dots grown from wurtzite CdSe cores still suffer from irreproducibility due to gel formation during the core synthesis. Moreover, state-of-the-art shelling procedures often remain time-consuming. We report here (1) a new procedure of the wurtzite CdSe quantum dot synthesis which eliminates the phosphonic anhydride contamination and (2) the improvement and generalization of the flash synthesis for the growth of highly luminescent multishell heterostructures. The high versatility of the flash procedure enables the epitaxial shelling of wurtzite CdSe by CdS, ZnS and even CdZnS alloys, resulting in a significant enhancement of their photoluminescence efficiency. Thanks to a modification of the CdSe/CdS dot-in-rods synthesis to prevent tip-to-tip connections, the flash procedure could also be extended to these anisotropic structures for the growth of multishell nanorods as well. The high quality optical properties of these multishell flash heterostructures are of great interest for applications ranging from lighting and displays to long-term cell labeling.