We previously reported a molecular hopper, which makes sub-nanometer steps by thiol-disulfide interchange along a track with cysteine footholds within a protein nanopore. Here we optimize the hopping rate (ca. 0.1 s-1 in the previous work) with a view towards rapid enzymeless biopolymer characterization during translocation within nanopores. We first took a single-molecule approach to obtain the reactivity profiles of individual footholds. The pK a values of cysteine thiols within a pore ranged from 9.17 to 9.85, and the pH-independent rate constants of the thiolates with a small-molecule disulfide varied by up to 20-fold. Through site-specific mutagenesis and a pH increase from 8.5 to 9.5, the overall hopping rate of a DNA cargo along a five-cysteine track was accelerated 4-fold, and the rate-limiting step 21-fold.
Chemical stepping of biopolymers within a protein nanopore suggests an enzymeless means of sequence characterization. Using a nanoreactor approach, we elucidated the reactivity profile of cysteine footholds along a protein track. Faster chemical stepping was achieved by rational mutagenesis and optimization of reaction conditions. In the future, the system has the potential for directional molecular motion at milliseconds per step.
Keywords: Mobile Molecules; Nanopores; Protein Engineering; Single-Molecule Chemistry; Thiol-Disulfide Interchange.
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