title: Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.
abstract: Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.
authors.affiliation: Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
authors.forename: Joongoo
authors.initials: J
authors.lastname: Lee
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Arnold J
authors.initials: AJ
authors.lastname: Boersma
authors.affiliation: Department of Chemistry, University of New Hampshire , Durham, New Hampshire 03824, United States.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Boudreau
authors.affiliation: Department of Pharmacology, University of Alberta , Edmonton, AB T6G 2R3, Canada.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cheley
authors.affiliation: Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
authors.forename: Oliver
authors.initials: O
authors.lastname: Daltrop
authors.affiliation: Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tamagaki
authors.affiliation: Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
keywords: membrane protein; nanoreactor; native chemical ligation; protein semisynthesis; single-molecule chemistry; unnatural amino acid