A study finds that human activity has led to a severe decrease in Caribbean shark abundance.
Image credit: Sean Mattson (International Center for Tropical Agriculture, Cali, Colombia).
Edited by Cynthia J. Burrows, University of Utah, Salt Lake City, UT, and approved September 16, 2015 (received for review June 2, 2015)
Significance
The modulation of ionic current flowing through an individual protein pore provides information at the single-molecule level about chemical reactions occurring within the pore. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. Here we have produced semisynthetic protein pores containing a terminal alkyne and used the pores as a nanoreactor to study Cu(I)-catalyzed azide-alkyne cycloaddition. A long-lived intermediate (4.5 s) in the reaction was directly observed.
Abstract
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 α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL 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.
Footnotes
- ↵1To whom correspondence should be addressed. Email: hagan.bayley{at}chem.ox.ac.uk.
Author contributions: J.L. and H.B. designed research; J.L. performed research; J.L. analyzed data; and J.L. and H.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1510565112/-/DCSupplemental.
Semisynthetic protein nanoreactor
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