Semisynthetic protein nanoreactor for single-molecule chemistry

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.semisynthesis | alpha-hemolysin | single-molecule chemistry | click chemistry

Engineered transmembrane pores

“…This approach has been further developed (by others) for expression in E. coli, but neither technique readily allows the production of proteins with multiple unnatural side chains. With that in mind, we have recently used native chemical ligation (NCL) to construct α-hemolysin polypeptides (Figure 1b) [29]. Solid-phase peptide synthesis was used to make the central segment of the 293-amino-acid polypeptide chain, which forms the transmembrane β barrel.…”

“…The full-length α-hemolysin polypeptide was obtained by ligation of the central peptide to flanking recombinant polypeptides. In an initial study, an amino acid with an alkyne side chain that projects into the lumen of the pore was used to investigate alkyne/azide click chemistry at the single-molecule level and revealed an intermediate with a mean lifetime of 4.5 s [29]. …”

“…An engineered pore with one such subunit was used as a nanoreactor to monitor alkyne/azide click chemistry at the single-molecule level. An intermediate (S 1 ) was revealed with a mean lifetime of 4.5 s [29]. …”

Bioorthogonal Cycloadditions with Sub‐Millisecond Intermediates

“…[6] In contrast, research into the dynamics of N 2 and CO 2 release,w hich yields the final products,has been largely limited to computational studies, [15] most likely because the intermediates are predicted to be too short-lived to be observed by approaches such as stopped-flow spectroscopy (ca. [17][18][19][20] Thetime resolution of the approach is limited by instrument noise to the microsecond domain. Indeed, quantum mechanical (QM) and quasi-classical trajectory (QCT) simulations with simplified reactants in the gas phase have suggested that the barrier to N 2 and CO 2 loss is low and that the bicyclic adducts have lifetimes on the order of aC ÀCbond vibration (ca.…”

“…[16] An internal residue within the transmembrane b barrel of the aHL pore is modified with ar eactant of interest. [17][18][19][20] Thetime resolution of the approach is limited by instrument noise to the microsecond domain. Cycloadditions that release N 2 or CO 2 .a )iEDDA reactions between atetrazine and astrained alkene or alkyne.F or the alkene, the subsequent 1,3-prototropic isomerisations tep leading to the 1,4dihydropyridazine is omitted here because the rate is too low for the isomerisation to be detected by the nanoreactora pproach.…”