Oxidative Folding of Conotoxins in Immobilized Systems

Green, Brad R.; Bułaj, Grzegorz

doi:10.2174/092986606774502162

Cited by 21 publications

(19 citation statements)

References 21 publications

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“…21 This polymer-supported oxidant had been previously applied to the oxidative folding of one-, two-, and three-disulfide peptides, 21,22 but to our knowledge is yet to be attempted for a tightly folded four-disulfide peptide such as hepcidin. Although it was unclear how the polymer-supported folding of a complicated four-disulfide peptide would proceed, we investigated the CLEAR-OX 2 -assisted folding of purified linear hepcidin at pH 3.3, 4.0, 5.5, and 7.5.…”

Section: Oxidative Folding Of Hepcidin At Acidic Ph 261mentioning

confidence: 99%

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Oxidative folding of hepcidin at acidic pH

et al. 2010

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Hepcidin is a four disulfide 25-residue peptide hormone which has a central role in the regulation of iron homeostasis. To support studies on hepcidin we have sought to establish reliable and robust synthetic methods for the preparation of correctly folded materials. While correctly-folded hepcidin has good aqueous solubility, we have found that its direct synthetic precursor, linear (reduced) hepcidin peptide, is resistant to solubilization, prone to precipitation at pH > or = 6, and thus difficult to fold efficiently. Attempts to directly fold either the crude or purified linear hepcidin peptide by air or DMSO oxidation methods under basic conditions were ineffective. However, addition of a glutathione redox pair system improved folding of purified linear hepcidin at mild basic pH (pH 7.5). Under acidic conditions, it was possible to oxidatively fold both crude and purified hepcidin using a polymer-supported oxidizing strategy. Peptide precipitation was also avoided under acidic conditions. Isolated folding yields of human hepcidin under acidic polymer-assisted conditions were superior to yields under basic folding conditions. These studies enabled identification of a reliable synthetic route for correctly-folded hepcidin.

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Section: Oxidative Folding Of Hepcidin At Acidic Ph 261mentioning

confidence: 99%

“…15 The activity of both the synthesized and urinary material were comparable (EC 50 ¼ 45 nM and *20 nM for the synthetic and urinary material, respectively) (see Figure 8). No activity was observed with [Abu 7,10,11,13,14,19,22,23 ] hepcidin, a cysteinefree linear analog of hepcidin.…”

Section: Assessment Of Human Hepcidin Disulfide Connectivity and In Vmentioning

confidence: 99%

Oxidative folding of hepcidin at acidic pH

et al. 2010

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“…All peptides, except AusB, were C -terminally amidated, purified by HPLC and analyzed with LC-MS, then freeze-dried and stored at −20 °C until use. The peptides were folded using an oxidative folding solution (1 mM reduced glutathione (Sigma, Munich, Germany), 1 mM oxidized glutathione (Roche, Mannheim, Germany), 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma, Munich, Germany) and 100 mM Tris/HCl (Merck, Darmstadt, Germany) [53]). The solution was adjusted to pH 7.63 with 10 M NaOH (Merck, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

Novel Conopeptides of Largely Unexplored Indo Pacific Conus sp.

et al. 2016

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Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage <50%). A third peptide, Asi3a from C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains.

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“…Significant efforts have been made to determine folding pathways and optimize oxidative folding conditions [129,131,[147][148][149][150][151][152][153][154].…”

Section: Oxidative Foldingmentioning

confidence: 99%

Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering

Avrutina

2016

Advances in Experimental Medicine and Biology

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Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.

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Oxidative Folding of Conotoxins in Immobilized Systems

Cited by 21 publications

References 21 publications

Oxidative folding of hepcidin at acidic pH

Oxidative folding of hepcidin at acidic pH

Novel Conopeptides of Largely Unexplored Indo Pacific Conus sp.

Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering

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