Prosequence-Mediated Disulfide Coupled Folding of the Peptide Hormones Guanylin and Uroguanylin

Lauber, Thomas; Marx, Ute C.

doi:10.2174/0929866053005836

Cited by 2 publications

(1 citation statement)

References 23 publications

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“…The existing experimental evidence on the oxidative folding of guanylin 67 and proguanylin 74 , 76 , 77 provides a good insight into the role of the prohormone. As it appears, the body of proguanylin folds first; the folded body provides a template for subsequent oxidative folding of the guanylin tail.…”

Section: Resultsmentioning

confidence: 99%

Simple MD-based model for oxidative folding of peptides and proteins

Izmailov

Podkorytov

Skrynnikov

2017

Sci Rep

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Significant strides have been recently made to fold peptides and small proteins in silico using MD simulations. However, facilities are currently lacking to include disulfide bonding in the MD models of protein folding. To address this problem, we have developed a simple empirical protocol to model formation of disulfides, which is perturbation-free, retains the same speed as conventional MD simulations and allows one to control the reaction rate. The new protocol has been tested on 15-aminoacid peptide guanylin containing four cysteine residues; the net simulation time using Amber ff14SB force field was 61 μs. The resulting isomer distribution is in qualitative agreement with experiment, suggesting that oxidative folding of guanylin in vitro occurs under kinetic control. The highly stable conformation of the so-called isomer 2(B) has been obtained for full-length guanylin, which is significantly different from the poorly ordered structure of the truncated peptide PDB ID 1GNB. In addition, we have simulated oxidative folding of guanylin within the 94-aminoacid prohormone proguanylin. The obtained structure is in good agreement with the NMR coordinates 1O8R. The proposed modeling strategy can help to explore certain fundamental aspects of protein folding and is potentially relevant for manufacturing of synthetic peptides and recombinant proteins.

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Section: Resultsmentioning

confidence: 99%

Simple MD-based model for oxidative folding of peptides and proteins

Izmailov

Podkorytov

Skrynnikov

2017

Sci Rep

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Prosequence switching: An effective strategy to produce biologically activeE. coliheat-stable enterotoxin STh

Weiglmeier

Berkner

Seebahn

et al. 2013

Journal of Biomolecular Structure and Dynamics

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Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.

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Prosequence-Mediated Disulfide Coupled Folding of the Peptide Hormones Guanylin and Uroguanylin

Cited by 2 publications

References 23 publications

Simple MD-based model for oxidative folding of peptides and proteins

Simple MD-based model for oxidative folding of peptides and proteins

Prosequence switching: An effective strategy to produce biologically activeE. coliheat-stable enterotoxin STh

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