The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the alpha-hemolysin (alpha HL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane beta-barrel of the assembled heptamer. The full-length alpha HL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. alpha HL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.
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