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Methods Mol Biol. 2013;966:325-36. doi: 10.1007/978-1-62703-245-2_20.

Protein disulfide bond formation in the periplasm: determination of the in vivo redox state of cysteine residues.

Author information

1
Brussels Center for Redox Biology and de Duve Institute, Université Catholique de Louvain, Brussels, Belgium.

Abstract

Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role, increasing protein stability, or reversibly form in the catalytic site of periplasmic oxidoreductases. Monitoring the in vivo redox state of cysteine residues, i.e., determining whether those cysteines are oxidized to a disulfide bond or not, is therefore required to fully characterize the function and structural properties of numerous periplasmic proteins. Here, we describe a reliable and rapid method based on trapping reduced cysteine residues with 4'-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), a maleimide compound. We use the Escherichia coli DsbA protein to illustrate the method, which can be applied to all envelope proteins.

PMID:
23299744
DOI:
10.1007/978-1-62703-245-2_20
[Indexed for MEDLINE]

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