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

Author:

Bo Zonghua1ORCID,Lim Zhong Hui1ORCID,Duarte Fernanda1ORCID,Bayley Hagan1ORCID,Qing Yujia1ORCID

Affiliation:

1. Department of Chemistry University of Oxford Mansfield Road Oxford OX1 3TA UK

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.

Funder

H2020 European Research Council

Oxford Nanopore Technologies

Wellcome Trust

Engineering and Physical Sciences Research Council

China Scholarship Council

Oxford University

Magdalen College, University of Oxford

Publisher

Wiley

Subject

General Medicine

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