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ACS Nano. 2016 Sep 27;10(9):8843-50. doi: 10.1021/acsnano.6b04663. Epub 2016 Aug 30.

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.

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Department of Chemistry, University of Oxford , Oxford OX1 3TA, U.K.
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , 9747 AG Groningen, The Netherlands.
Department of Chemistry, University of New Hampshire , Durham, New Hampshire 03824, United States.
Department of Pharmacology, University of Alberta , Edmonton, AB T6G 2R3, Canada.


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.


membrane protein; nanoreactor; native chemical ligation; protein semisynthesis; single-molecule chemistry; unnatural amino acid

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