title: Intramolecular disulfide bond between catalytic cysteines in an intein precursor.
abstract: Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic  Pyrococcus abyssi  PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.forename: Lingyun
authors.initials: L
authors.lastname: Li
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Du
authors.forename: Jiajing
authors.initials: J
authors.lastname: Liu
authors.forename: Julie N
authors.initials: JN
authors.lastname: Reitter
authors.forename: Kenneth V
authors.initials: KV
authors.lastname: Mills
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
mesh_terms: D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D047668:Inteins; D043903:Pyrococcus abyssi / Q000737:chemistry
chemical_list: D004220:Disulfides; D003545:Cysteine