Determination of Amide cis/trans Isomers in N-Acetyl-d-glucosamine: Tailored NMR Analysis of the N-Acetyl Group Conformation

N-Acetyl-d-glucosamine (GlcNAc) is one of the most common amino sugars in nature, but the conformation of its N-acetyl group has drawn little attention. We report herein the first identification of NH protons of the amide cis forms of alpha- and beta-GlcNAc by NMR spectroscopy. Relative quantification and thermodynamic analysis of both cis and trans forms was carried out in aqueous solution. The NH protons were further utilized by adapting protein NMR experiments to measure eight J-couplings within the N-acetyl group, of which six are sensitive to the H2-NH conformation and two are sensitive to the amide conformation. For amide cis and trans forms, the orientation between H2 and NH was determined as anti conformation, while a small percentage of syn conformation was predicted for the amide trans form of beta-GlcNAc. This approach holds great promise for the detailed conformational analysis of GlcNAc in larger biomolecules, such as glycoproteins and polysaccharides

Carbohydrate and metabonomic studies by NMR spectroscopy

NMR spectroscopy can be used to investigate organic and biomolecular structures, both as pure compounds and in complex mixtures. In this thesis NMR spectroscopy was used for carbohydrate and metabonomic studies. Lipopolysaccharides are major components of the outer cell membrane of Gram-negative bacteria and are involved in pathogenic interactions. The structure of a lipopolysaccharide from the human pathogen Plesiomonas shigelloides was analyzed by NMR spectroscopy and mass spectrometry. The structure contained an O-specific polysaccharide with a tetrasaccharide repeating unit. The core oligosaccharide was an undecasaccharide with previously not reported heterogeneity within the carbohydrate backbone. Hyaluronan is a glycosaminoglycan with high viscosity and water-retaining ability. Hydroxy protons of hyaluronan oligosaccharides were studied by NMR spectroscopy in aqueous solution to investigate hydrogen bonding interactions and hydration. Weak hydrogen bonding was observed between hydroxy protons and the ring oxygens over the β(1→3) and β(1→4) glycosidic linkages. A chemical exchange interaction was also identified between O(4)H of N-acetylglucosamine and O(3)H of glucuronic acid across the β(1→3) linkage. The interaction could be mediated through water bridges and thus contribute to the water-retaining ability of hyaluronan. Polyunsaturated fatty acids are essential nutritional components of marine lipid sources. A ¹H HR-MAS NMR method was developed to determine the amount of essential polyunsaturated fatty acids in fish muscle without any pretreatment. Additionally, the small metabolite profile of the fish muscle was obtained, which can be used to determine fish quality. Caloric restriction is known to increase the lifespan of rats. In order to evaluate the metabolic responses to graded caloric restriction in rats, blood serum was analyzed by NMR spectroscopy. Multivariate analysis showed a decrease in blood lipids and alanine, and an increase in creatine and 3-hydroxybutyrate as a response to caloric restriction

Band-selective NMR experiments for suppression of unwanted signals in complex mixtures

Band-selective NMR experiments are presented that allow selective suppression of unwanted signals (SUN) from the spectra of complex metabolite mixtures. As a result, spectral overlap and dynamic range problems are substantially reduced and low-intensity signals normally covered by dominant signals can be observed. The usefulness of the experiments is exemplified with selective suppression of sugar signals from the NMR spectra of fruit juice and a plant sample. Other possible applications include blood, milk, and wine samples

Factors influencing stoichiometry and stability of polyoxometalate - peptide complexes

In the pursuit of understanding the factors guiding interactions between polyoxometalates (POMs) and biomolecules, several complexes between Keggin phosphomolybdate and diglycine have been produced at different acidity and salinity conditions, leading to difference in stoichiometry and in crystal structure. Principal factors determining how the POM and dipeptide interact appear to be pH, ionic strength of the medium, and the molar ratio of POM to peptide. An important effect turned out to be even the structure-directing role of the sodium cations coordinating carbonyl functions of the peptide bond. Given the interest in applying POMs in biological systems, these factors are highly relevant to consider. In the view of recent interest in using POMs as nano catalysts in peptide hydrolysis also the potential Keggin POM transformation in phosphate buffered saline medium was investigated leading to insight that nanoparticles of zirconium phosphate (ZrP) can be actual catalysts for breakdown of the peptide bond

Extended automated quantification algorithm (AQuA) for targeted 1H NMR metabolomics of highly complex samples: application to plant root exudates

Introduction The Automated Quantification Algorithm (AQuA) is a rapid and efficient method for targeted NMR-based metabolomics, currently optimised for blood plasma. AQuA quantifies metabolites from 1D-H-1 NMR spectra based on the height of only one signal per metabolite, which minimises the computational time and workload of the method without compromising the quantification accuracy.Objectives To develop a fast and computationally efficient extension of AQuA for quantification of selected metabolites in highly complex samples, with minimal prior sample preparation. In particular, the method should be capable of handling interferences caused by broad background signals.Methods An automatic baseline correction function was combined with AQuA into an automated workflow, the extended AQuA, for quantification of metabolites in plant root exudate NMR spectra that contained broad background signals and baseline distortions. The approach was evaluated using simulations as well as a spike-in experiment in which known metabolite amounts were added to a complex sample matrix.Results The extended AQuA enables accurate quantification of metabolites in 1D-H-1 NMR spectra with varying complexity. The method is very fast (< 1 s per spectrum) and can be fully automated.Conclusions The extended AQuA is an automated quantification method intended for 1D-H-1 NMR spectra containing broad background signals and baseline distortions. Although the method was developed for plant root exudates, it should be readily applicable to any NMR spectra displaying similar issues as it is purely computational and applied to NMR spectra post-acquisition

A detailed picture of a protein–carbohydrate hydrogen-bonding network revealed by NMR and MD simulations

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents

Modulating Surface Properties of the Linothele fallax Spider Web by Solvent Treatment

[Image: see text] Linothele fallax (Mello-Leitão) (L. fallax) spider web, a potentially attractive tissue engineering material, was investigated using quantitative peak force measurement atomic force microscopy and scanning electron microscopy with energy dispersive spectroscopy both in its natural state and after treatment with solvents of different protein affinities, namely, water, ethanol, and dimethyl sulfoxide (DMSO). Native L. fallax silk threads are densely covered by globular objects, which constitute their inseparable parts. Depending on the solvent, treating L. fallax modifies its appearance. In the case of water and ethanol, the changes are minor. In contrast, DMSO practically removes the globules and fuses the threads into dense bands. Moreover, the solvent treatment influences the chemistry of the threads’ surface, changing their adhesive and, therefore, biocompatibility and cell adhesion properties. On the other hand, the solvent-treated web materials’ contact effect on different types of biological matter differs considerably. Protein-rich matter controls humidity better when wrapped in spider silk treated with more hydrophobic solvents. However, carbohydrate plant materials retain more moisture when wrapped in native spider silk. The extracts produced with the solvents were analyzed using nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry techniques, revealing unsaturated fatty acids as representative adsorbed species, which may explain the mild antibacterial effect of the spider silk. The extracted metabolites were similar for the different solvents, meaning that the globules were not “dissolved” but “fused into” the threads themselves, being supposedly rolled-in knots of the protein chain

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.EEA BalcarceFil: Zhao, Xue. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Jayarathna, Shishanthi. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Nestor, Gustav. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Beganovic, Mirela. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Andersson, Roger. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia

Complete H-1 and C-13 NMR spectral assignment of D-glucofuranose

Although D-glucose is the most common sugar in nature, only a few NMR studies have focused on its minor furanose forms, and they have been limited to the anomeric position. Here, complete H-1 and C-13 NMR spectral analysis of alpha- and beta-D-glucofuranose was performed, including signal assignment, chemical shifts, and coupling constants. Selective and non-selective 1D and 2D NMR experiments were used for the analysis, complemented by spin simulations and iterative spectral analysis

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans