12 research outputs found
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. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa
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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. Emerging strategies to allow direct detection of hydrogen bonds through heteronuclear couplings offer promise for the future. Molecular dynamic simulations are important in this effort both to predict and confirm hydrogen bond donors and acceptors
<sup>1</sup>H and <sup>15</sup>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 <i>N</i>-acetylated
and <i>N</i>-sulfated forms, and as the unmodified amine.
Though the <sup>1</sup>H and <sup>15</sup>N NMR chemical shifts of <i>N</i>-acetyl- and <i>N</i>-sulfoglucosamine residues
have been extensively characterized, this study provides the first
direct NMR characterization of the amine groups of glucosamine and
3-<i>O</i>-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-<i>O</i>-sulfoglucosamine is explored through <sup>1</sup>H NMR
p<i>K</i><sub>a</sub> measurements but is not supported
by the experimental results
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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. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa
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 (NHSO<sub>3</sub><sup>–</sup>) proton and the adjacent 3-<i>O</i>-sulfo group of the 3,6-<i>O</i>-sulfated <i>N</i>-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-<i>O</i>-sulfo-iduronic acid residues. NMR measurements
of temperature coefficients, chemical shift differences, and solvent
exchange rate constants provide experimental confirmation of these
hydrogen bonds. We note that the extraction of rate constants from
cross-peak buildup curves in 2D exchange spectroscopy is complicated
by the presence of radiation damping in aqueous solution. A straightforward
model is presented that explicitly takes into account the effects
of radiation damping on the water proton relaxation and is sufficiently
robust to provide an accurate measure of the proton exchange rate
between the analyte hydroxyl protons and water
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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. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases
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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-ICVWQDWÂGAHRCT-NH<sub>2</sub>, cyclized
between C2 and C12), based on structural, computational, and experimental
studies. Furthermore, we developed and utilized a computational framework
for the design of peptides containing non-natural amino acids. These
new compstatin peptides contain polar N-terminal extensions and non-natural
amino acid substitutions at positions 4 and 9. Peptides with α-modified
non-natural alanine analogs at position 9, as well as peptides containing
only N-terminal polar extensions, exhibited similar activity compared
to W4A9, as quantified via ELISA, hemolytic, and cell-based assays,
and showed improved solubility, as measured by UV absorbance and reverse-phase
HPLC experiments. Because of their potency and solubility, these peptides
are promising candidates for therapeutic development in numerous complement-mediated
diseases