Mobile Molecules: Reactivity Profiling Guides Faster Movement on a Cysteine Track

Abstract

AbstractWe 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 pKa 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.