ResearchPad - nmr-spectroscopy Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[NMR resonance assignment and structure prediction of the C-terminal domain of the microtubule end-binding protein 3]]> End-binding proteins (EBs) associate with the growing microtubule plus ends to regulate microtubule dynamics as well as the interaction with intracellular structures. EB3 contributes to pathological vascular leakage through interacting with the inositol 1,4,5-trisphosphate receptor 3 (IP3R3), a calcium channel located at the endoplasmic reticulum membrane. The C-terminal domain of EB3 (residues 200–281) is functionally important for this interaction because it contains the effector binding sites, a prerequisite for EB3 activity and specificity. Structural data for this domain is limited. Here, we report the backbone chemical shift assignments for the human EB3 C-terminal domain and computationally explore its EB3 conformations. Backbone assignments, along with computational models, will allow future investigation of EB3 structural dynamics, interactions with effectors, and will facilitate the development of novel EB3 inhibitors.

<![CDATA[Bioconversion of fructus sophorae into 5,7,8,4’-tetrahydroxyis oflavone with Aspergillus aculeatus]]>

A fungus identified as Aspergillus aculeatus was used to biotransform genistein and glycosides to polyhydroxylated isoflavones. The strain was identified on the basis of colony morphology features and ITS rDNA sequence analysis. Phylogenetic tree was constructed to determine its taxonomic status. Genistein and glycosides were transformed by Aspergillus aculeatus to 5,7,8,4’- tetrahydroxyisoflavone. The chemical structure of the product was identified by high performance liquid chromatography(HPLC), liquid chromatography-mass spectrometry(LC/MS), Infrared spectroscopy (IR) and NMR spectrometer methods. The ITS rDNA sequence of the strain had 100% similarity with Aspergillus. Furthermore, it was ultimately identified as Aspergillus aculeatus. The metabolite of genistein and glycosides was identified as 5,7,8,4’-tetrahydroxyisoflavone. 120 mg 5,7,8,4’-tetrahydroxyisoflavone was made from 20 g fructus sophorae, which was bioconverted unconditionally by Aspergillus aculeatus for 96 h, and the purity was 96%. On the basis of the findings, Aspergillus aculeatus was a novel strain with specific ability to convert genistein and glycosides into 5,7,8,4’-tetrahydroxyisoflavone which had potential applications.

<![CDATA[S100A4 inhibits cell proliferation by interfering with the S100A1-RAGE V domain]]>

The Ca2+-dependent human S100A4 (Mts1) protein is part of the S100 family. Here, we studied the interactions of S100A4 with S100A1 using nuclear magnetic resonance (NMR) spectroscopy. We used the chemical shift perturbed residues from HSQC to model S100A4 and S100A1 complex with HADDOCK software. We observed that S100A1 and the RAGE V domain have an analogous binding area in S100A4. We discovered that S100A4 acts as an antagonist among the RAGE V domain and S100A1, which inhibits tumorigenesis and cell proliferation. We used a WST-1 assay to examine the bioactivity of S100A1 and S100A4. This study could possibly be beneficial for evaluating new proteins for the treatment of diseases.

<![CDATA[Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds]]>

Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2’s catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG) and maximal rate constant (k’ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of ‘kinetic’ disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.

<![CDATA[Heat stress modifies the lactational performances and the urinary metabolomic profile related to gastrointestinal microbiota of dairy goats]]>

The aim of the study is to identify the candidate biomarkers of heat stress (HS) in the urine of lactating dairy goats through the application of proton Nuclear Magnetic Resonance (1H NMR)-based metabolomic analysis. Dairy does (n = 16) in mid-lactation were submitted to thermal neutral (TN; indoors; 15 to 20°C; 40 to 45% humidity) or HS (climatic chamber; 37°C day, 30°C night; 40% humidity) conditions according to a crossover design (2 periods of 21 days). Thermophysiological traits and lactational performances were recorded and milk composition analyzed during each period. Urine samples were collected at day 15 of each period for 1H NMR spectroscopy analysis. Principal component analysis (PCA) and partial least square—discriminant analysis (PLS-DA) assessment with cross validation were used to identify the goat urinary metabolome from the Human Metabolome Data Base. HS increased rectal temperature (1.2°C), respiratory rate (3.5-fold) and water intake (74%), but decreased feed intake (35%) and body weight (5%) of the lactating does. No differences were detected in milk yield, but HS decreased the milk contents of fat (9%), protein (16%) and lactose (5%). Metabolomics allowed separating TN and HS urinary clusters by PLS-DA. Most discriminating metabolites were hippurate and other phenylalanine (Phe) derivative compounds, which increased in HS vs. TN does. The greater excretion of these gut-derived toxic compounds indicated that HS induced a harmful gastrointestinal microbiota overgrowth, which should have sequestered aromatic amino acids for their metabolism and decreased the synthesis of neurotransmitters and thyroid hormones, with a negative impact on milk yield and composition. In conclusion, HS markedly changed the thermophysiological traits and lactational performances of dairy goats, which were translated into their urinary metabolomic profile through the presence of gut-derived toxic compounds. Hippurate and other Phe-derivative compounds are suggested as urinary biomarkers to detect heat-stressed dairy animals in practice.

<![CDATA[Gray matter atrophy in multiple sclerosis despite clinical and lesion stability during natalizumab treatment]]>


Brain volume loss is an important surrogate marker for assessing disability in MS; however, contribution of gray and white matter to the whole brain volume loss needs further examination in the context of specific MS treatment.


To examine whole and segmented gray, white, thalamic, and corpus callosum volume loss in stable patients receiving natalizumab for 2–5 years.


This was a retrospective study of 20 patients undergoing treatment with natalizumab for 24–68 months. Whole brain volume loss was determined with SIENA. Gray and white matter segmentation was done using FAST. Thalamic and corpus callosum volumes were determined using Freesurfer. T1 relaxation values of chronic hypointense lesions (black holes) were determined using a quantitative, in-house developed method to assess lesion evolution.


Over a mean of 36.6 months, median percent brain volume change (PBVC) was -2.0% (IQR 0.99–2.99). There was decline in gray (p = 0.001) but not white matter (p = 0.6), and thalamic (p = 0.01) but not corpus callosum volume (p = 0.09). Gray matter loss correlated with PBVC (Spearman’s r = 0.64, p = 0.003) but not white matter (Spearman’s r = 0.42, p = 0.07). Age significantly influenced whole brain volume loss (p = 0.010, multivariate regression), but disease duration and baseline T2 lesion volume did not. There was no change in T1 relaxation values of lesions or T2 lesion volume over time. All patients remained clinically stable.


These results demonstrate that brain volume loss in MS is primarily driven by gray matter changes and may be independent of clinically effective treatment.

<![CDATA[Characterization of the antibacterial activity of Bald’s eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa]]>

Bald’s eyesalve is an Anglo-Saxon medicinal remedy that has been used through ancient times to treat eye sty infections and may represent a source of ancientbiotics. This study assessed the efficacy of Bald’s eyesalve against several strains of Staphylococcus aureus and Pseudomonas aeruginosa, including a multi-drug resistant phenotype, and identified the principal compound conveying antibacterial activity. Bald’s eyesalve formulations were produced by combining garlic, onion or leek, wine, bovine bile, and brass, with specific ingredient omissions in several formulations, followed by incubation at 4 °C for 9 days. Bald’s eyesalve formulation ES-GBBr exhibited the greatest antibacterial activity against S. aureus and P. aeruginosa. Fractionation of ES-GBBr using molecular size exclusion and organic solvent partitioning isolated its antibacterial activity to the small molecule nonpolar fraction, and 1D 1H NMR revealed the identity of the antibacterial agent to be allicin. Depletion of allicin from this fraction by addition of exogenous cysteine established that all observable growth inhibition originated from allicin. Quantification of allicin demonstrated that its concentration was significantly greater in ES-GBBr compared to the ES-O formulation; however, this was not due to greater yield. The antibacterial activity of allicin against S. aureus was antagonized by other ingredients within Bald’s eyesalve, whereas they were additive or synergistic against P. aeruginosa. These results suggest that neither leek nor onion is necessary for the antibacterial efficacy of Bald’s eyesalve against S. aureus or P. aeruginosa, and while allicin was identified as the principal antibacterial agent present, its activity is influenced differentially in the presence of additional Bald’s eyesalve ingredients when used against S. aureus compared to P. aeruginosa. Ancientbiotics may provide a source of promising antibacterials; however, identifying the source of activity and assessing distinct formulations for cooperative effects are essential to using ancient remedies, such as Bald’s eyesalve, effectively against drug resistant pathogens.

<![CDATA[A set of conformationally well-defined L/D-peptide epitopes provides a serological bar code for autoantibody subtypes]]>

Which conformational parameters lead to an antibody-affine peptide antigen? And in how many different conformations can we actually present the respective conformational epitope? To provide answers from a chemical point of view, we direct the bending and tethering of peptide backbones by the utilisation of a hydrophobic cluster, disulfides, and d-amino acids. Each mutation is employed pairwise on directly opposite sides of a β-hairpin. In combination, these synthetic modules guide the formation of complementary β-sheet-like structures, whereby the oppositely configured (l/d-)bi-disulfide pairs form with high regioselectivity. The conformational properties of the peptides are assessed by NMR spectroscopy and correlated with their antibody affinity in ELISA. From a pool of thus designed peptide antigens with distinctive complementary affinities against known rheumatoid arthritis (RA) autoantibodies, we select a set of epitopes for an immunoassay with sera of RA patients. We want to put emphasis on the idea, that the different conformational properties of the chosen antigens, containing the same epitope sequence, are mirrored in the distribution of autoantibody subtypes (or of the antibody polyclonality, respectively). Such directly comparable information can only be delivered by a set of peptides, rather than a single one. The hairpin-restriction technology of l/d-configured bi-disulfide amino acid pairs is not limited to RA but applicable to other shape-persistent hairpin motifs which are supposed to identify subgroups of protein receptors.

<![CDATA[Metabolic fingerprint of insulin resistance in human polymorphonuclear leucocytes]]>

The present study was aimed at determining the metabolic profile of PMNs in obese subjects, and to explore its potential relationship with insulin resistance (IR). To achieve this goal, a pilot clinical study was performed using PMNs from 17 patients with obesity and IR, and 17 lean controls without IR, which was validated in an additional smaller cohort (consisting of 10 patients and 10 controls). PMNs were isolated from peripheral blood and nuclear magnetic resonance was used to perform the metabolomic analysis. A total of 48 metabolites were quantified. The main metabolic change found in PMNs was a significant increase in 2-aminoisobutyric acid with a direct correlation with HOMA-IR (p<0.001), BMI (p<0.000001) and waist circumference (p<0.000001). By contrast, a decrease of 3-hydroxyisovalerate was observed with an inverse correlation with HOMA-IR (p = 0.001), BMI (p = 0.001) and waist circumference (p = 0.0001). Notably, the metabolic profile in plasma was different than that obtained in PMNs. In summary, our results suggest that the change in 3-hydroxyisovalerate and 2-aminoisobutyric is the key metabolic fingerprint in PMNs of obese subjects with IR. In addition, our methodology could be an easy and reliable tool for monitoring the effect of treatments in the setting of precision medicine.

<![CDATA[Evidence of cyclical light/dark-regulated expression of freezing tolerance in young winter wheat plants]]>

The ability of winter wheat (Triticum aestivum L.) plants to develop freezing tolerance through cold acclimation is a complex rait that responds to many environmental cues including day length and temperature. A large part of the freezing tolerance is conditioned by the C-repeat binding factor (CBF) gene regulon. We investigated whether the level of freezing tolerance of 12 winter wheat lines varied throughout the day and night in plants grown under a constant low temperature and a 12-hour photoperiod. Freezing tolerance was significantly greater (P<0.0001) when exposure to subfreezing temperatures began at the midpoint of the light period, or the midpoint of the dark period, compared to the end of either period, with an average of 21.3% improvement in survival. Thus, freezing survival was related to the photoperiod, but cycled from low, to high, to low within each 12-hour light period and within each 12-hour dark period, indicating ultradian cyclic variation of freezing tolerance. Quantitative real-time PCR analysis of expression levels of CBF genes 14 and 15 indicated that expression of these two genes also varied cyclically, but essentially 180° out of phase with each other. Proton nuclear magnetic resonance analysis (1H-NMR) showed that the chemical composition of the wheat plants' cellular fluid varied diurnally, with consistent separation of the light and dark phases of growth. A compound identified as glutamine was consistently found in greater concentration in a strongly freezing-tolerant wheat line, compared to moderately and poorly freezing-tolerant lines. The glutamine also varied in ultradian fashion in the freezing-tolerant wheat line, consistent with the ultradian variation in freezing tolerance, but did not vary in the less-tolerant lines. These results suggest at least two distinct signaling pathways, one conditioning freezing tolerance in the light, and one conditioning freezing tolerance in the dark; both are at least partially under the control of the CBF regulon.

<![CDATA[Chemical structures and characteristics of animal manures and composts during composting and assessment of maturity indices]]>

Changes in physicochemical characteristics, chemical structures and maturity of swine, cattle and chicken manures and composts during 70-day composting without addition of bulking agents were investigated. Physicochemical characteristics were measured by routine analyses and chemical structures by solid-state 13C NMR and FT-IR. Three manures were of distinct properties. Their changes in physicochemical characteristics, chemical structures, and maturity were different not only from each other but also from those with addition of bulking agents during composting. Aromaticity in chicken manure composts decreased at first, and then increased whereas that in cattle and swine manure composts increased. Enhanced ammonia volatilization occurred without addition of bulking agents. NMR structural information indicated that cattle and chicken composts were relatively stable at day 36 and 56, respectively, but swine manure composts were not mature up to day 70. Finally, the days required for three manures to reach the threshold values of different maturity indices were different.

<![CDATA[The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells]]>

Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine. We have identified this compound in three independent cyanobacterial strains of genera Nostoc, Nodularia, and Anabaena and recognized the putative gene clusters for NoA biosynthesis in their genomes. Its structure was characterized using a combination of NMR, HRMS and FTIR methods. The compound was first isolated as a positive hit during screening for apoptotic inducers in crude cyanobacterial extracts. We demonstrated that NoA-induced cell death has attributes of caspase-dependent apoptosis. Moreover, NoA exhibits a potent anti-proliferative activity (0.7–4.5 μM) against several human cancer lines, with p53-mutated cell lines being even more sensitive. Since cancers bearing p53 mutations are resistant to several conventional anti-cancer drugs, NoA may offer a new scaffold for the development of drugs that have the potential to target tumor cells independent of their p53 status. As no analogous type of compound was previously described in the nature, NoA establishes a novel class of bioactive secondary metabolites.

<![CDATA[Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA]]>

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.

<![CDATA[The Role of High-Resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy for Predicting the Invasive Component in Patients with Ductal Carcinoma In Situ Diagnosed on Preoperative Biopsy]]>

The purpose of this study was to evaluate the role of high-resolution magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy in patients with ductal carcinoma in situ (DCIS) diagnosed on preoperative biopsy. We investigated whether the metabolic profiling of tissue samples using HR-MAS 1H NMR spectroscopy could be used to distinguish between DCIS lesions with or without an invasive component. Our institutional review board approved this combined retrospective and prospective study. Tissue samples were collected from 30 patients with pure DCIS and from 30 with DCIS accompanying invasive carcinoma. All patients were diagnosed with DCIS by preoperative core-needle biopsy and underwent surgical resection. The metabolic profiling of tissue samples was performed by HR-MAS 1H NMR spectroscopy. All observable metabolite signals were identified and quantified in all tissue samples. Metabolite intensity normalized by total spectral intensities was compared according to the tumor type using the Mann-Whitney test. Multivariate analysis was performed with orthogonal projections to latent structure-discriminant analysis (OPLS-DA). By univariate analysis, the metabolite concentrations of choline-containing compounds obtained with HR-MAS 1H NMR spectroscopy did not differ significantly between the pure DCIS and DCIS accompanying invasive carcinoma groups. However, the GPC/PC ratio was higher in the pure DCIS group than in the DCIS accompanying invasive carcinoma group (p = 0.004, Bonferroni-corrected p = 0.064), as well as the concentration of myo-inositol and succinate. By multivariate analysis, the OPLS-DA models built with HR-MAS MR metabolic profiles could clearly discriminate between pure DCIS and DCIS accompanying invasive carcinoma. Our preliminary results suggest that HR-MAS MR metabolomics on breast tissue may be able to distinguish between DCIS lesions with or without an invasive component.

<![CDATA[Discrimination of Basal Cell Carcinoma from Normal Skin Tissue Using High-Resolution Magic Angle Spinning 1H NMR Spectroscopy]]>

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy is a useful tool for investigating the metabolism of various cancers. Basal cell carcinoma (BCC) is the most common skin cancer. However, to our knowledge, data on metabolic profiling of BCC have not been reported in the literature. The objective of the present study was to investigate the metabolic profiling of cutaneous BCC using HR-MAS 1H NMR spectroscopy. HR-MAS 1H NMR spectroscopy was used to analyze the metabolite profile and metabolite intensity of histopathologically confirmed BCC tissues and normal skin tissue (NST) samples. The metabolic intensity normalized to the total spectral intensities in BCC and NST was compared, and multivariate analysis was performed with orthogonal partial least-squares discriminant analysis (OPLS-DA). P values < 0.05 were considered statistically significant. Univariate analysis revealed 9 metabolites that showed statistically significant difference between BCC and NST. In multivariate analysis, the OPLS-DA models built with the HR-MAS NMR metabolic profiles revealed a clear separation of BCC from NST. The receiver operating characteristic curve generated from the results revealed an excellent discrimination of BCC from NST with an area under the curve (AUC) value of 0.961. The present study demonstrated that the metabolite profile and metabolite intensity differ between BCC and NST, and that HR-MAS 1H NMR spectroscopy can be a valuable tool in the diagnosis of BCC.

<![CDATA[Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3]]>

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

<![CDATA[8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme]]>

8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface.

<![CDATA[Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core]]>

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

<![CDATA[Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1]]>

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.

<![CDATA[Thermodynamic Features of Structural Motifs Formed by β-L-RNA]]>

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by 1H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.