Methods for Measuring Exchangeable Protons in Glycosaminoglycans.

Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra.

Screening enoxaparin tetrasaccharide SEC fractions for 3-O-sulfo-N-sulfoglucosamine residues using [(1)H,(15)N] HSQC NMR.

Heparin and heparan sulfate (HS) are important in mediating a variety of biological processes through binding to myriad different proteins. Specific structural elements along the polysaccharide chains are essential for high affinity protein binding, such as the 3-O-sulfated N-sulfoglucosamine (GlcNS3S) residue, a relatively rare modification essential for heparin's anticoagulant activity. The isolation of 3-O-sulfated oligosaccharides from complex mixtures is challenging because of their low abundance.

Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.

The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N.

(1)H and (15)N NMR Characterization of the Amine Groups of Heparan Sulfate Related Glucosamine Monosaccharides in Aqueous Solution.

Glucosamine is an important constituent of the heterogeneous glycosaminoglycans heparin and heparan sulfate occurring in N-acetylated and N-sulfated forms, and as the unmodified amine. Though the (1)H and (15)N NMR chemical shifts of N-acetyl- and N-sulfoglucosamine residues have been extensively characterized, this study provides the first direct NMR characterization of the amine groups of glucosamine and 3-O-sulfoglucosamine in aqueous solution. The solvent exchange properties of the amine protons are examined, and the possibility of a salt bridge between the sulfate and amine groups of 3-O-sulfoglucosamine is explored through (1)H NMR pKa measurements but is not supported by the experimental results.

New compstatin peptides containing N-terminal extensions and non-natural amino acids exhibit potent complement inhibition and improved solubility characteristics.

Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies.

Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface.

Methods for measuring exchangeable protons in glycosaminoglycans.

Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds, providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra.

Hydroxyl-proton hydrogen bonding in the heparin oligosaccharide Arixtra in aqueous solution.

Heparin is best known for its anticoagulant activity, which is mediated by the binding of a specific pentasaccharide sequence to the protease inhibitor antithrombin-III (AT-III). Although heparin oligosaccharides are thought to be flexible in aqueous solution, the recent discovery of a hydrogen bond between the sulfamate (NHSO3(-)) proton and the adjacent 3-O-sulfo group of the 3,6-O-sulfated N-sulfoglucosamine residue of the Arixtra (fondaparinux sodium) pentasaccharide demonstrates that definable elements of local structure are accessed. Molecular dynamics simulations of Arixtra suggest the presence of additional hydrogen bonds involving the C3-OH groups of the glucuronic acid and 2-O-sulfo-iduronic acid residues.

The interaction of enoxaparin and fondaparinux with calcium.

The main sites of calcium binding were determined for the low molecular weight heparin drug enoxaparin and the synthetic pentasaccharide Arixtra (fondaparinux). [(1)H,(13)C] HSQC pH titrations were carried out to characterize the acid-base properties of these samples both in the presence and absence of calcium. The differences in the titration curves were used to determine the structural components of enoxaparin and fondaparinux responsible for Ca(2+) binding.

Characterizing the microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1H and 15N NMR chemical shift analysis.

Heparin and heparan sulfate (HS) are members of a biologically important group of highly anionic linear polysaccharides called glycosaminoglycans (GAGs). Because of their structural complexity, the molecular-level characterization of heparin and HS continues to be a challenge. The work presented herein describes an emerging approach for the analysis of unfractionated and low molecular weight heparins, as well as porcine and human-derived HS.

Sulfamate proton solvent exchange in heparin oligosaccharides: evidence for a persistent hydrogen bond in the antithrombin-binding pentasaccharide Arixtra.

Sulfamate groups (NHSO(3)(-)) are important structural elements in the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). In this work, proton nuclear magnetic resonance (NMR) line-shape analysis is used to explore the solvent exchange properties of the sulfamate NH groups within heparin-related mono-, di-, tetra- and pentasaccharides as a function of pH and temperature. The results of these experiments identified a persistent hydrogen bond within the Arixtra (fondaparinux sodium) pentasaccharide between the internal glucosamine sulfamate NH and the adjacent 3-O-sulfo group.