Semisynthetic protein nanoreactor for single-molecule chemistry.
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.
Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom.
Joongoo
J
Lee
Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom hagan.bayley@chem.ox.ac.uk.
Hagan
H
Bayley
D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D057930:Click Chemistry / Q000379:methods*; D003300:Copper; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D036103:Nanotechnology / Q000379:methods*; D018272:Porins / Q000096:biosynthesis* / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*
D000480:Alkynes; D001386:Azides; D017931:DNA Primers; D018272:Porins; D003300:Copper
alpha-hemolysin; click chemistry; semisynthesis; single-molecule chemistry