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Antioxid Redox Signal. 2015 Oct 20;23(12):945-57. doi: 10.1089/ars.2014.6235. Epub 2015 Jul 20.

Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase.

Author information

1
1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
2
2 Actelion, Allschwil, Switzerland .
3
3 Department of Microbiology and Immunobiology, Harvard Medical School , Boston, Massachusetts.
4
4 Novartis, Basel, Switzerland .
5
5 Howard Hughes Medical Institute Molecular, Cellular and Developmental Biology, University of Michigan , Ann Arbor, Michigan.

Abstract

AIMS:

Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state.

RESULTS:

We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG.

INNOVATION AND CONCLUSIONS:

Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization.

PMID:
26191605
PMCID:
PMC4624244
DOI:
10.1089/ars.2014.6235
[Indexed for MEDLINE]
Free PMC Article

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