The role of the constriction in defining the molecular sensing trap of aerolysin-based pores.

Abstract

It has recently been shown that ionic current modulation by analytes visiting the pore formed by wild-type or mutant aerolysin (AeL) toxin is sensitive not only to molecular volume but also to molecular shape (1). One prerequisite of the remarkable sensitivity of this “whole molecule sensing” regime is the prolonged (1-100 ms) trapping of single molecules in the beta-barrel of the pore. At the same time, it is well known that macromolecular analytes are unable to enter this portion of the AeL-pore directly from the trans-side (2) but must enter from cis passinga constriction. We use high-bandwidth (0-100 kHz) recording of blockades induced by short polynucleotides (dA3,dA4,dA5) and oligoarginine peptides (R3,R5,R7) in conjunction with mutations of charged residues defining the trans-ward boundary of the constriction (D222C, R220S) to probe its role in the sensing mechanism. Both mutations induced rectification (D222C cis-ward; R220S trans-ward) and significantly affected the probability of transient deep blocks indicating analyte returns to the constriction after the first visit to the trap, with D222C potentiating and R220S reducing it for nuleotides. However, the initial deep block indicative of constriction passage remained in R220S. In the case of peptides, however, D222C left deep blocks largely unaffected while R220S nearly abolished returns and prolonged total blockage durations. These and other results indicate that the residues at the border between the constriction and the trap do not merely act by their charges but exert important steric influences and that further modifications of these residues may tailor the pore to various analytes. (1) Ouldali et al. 2020. Nat. Biotech. 38:176-181; Ensslen et al. 2022. JACS 144:16060-16068. (2) Baaken et al. 2015. ACS Nano. 9:6443-6449. (3) Iacovache et al. 2016. Nat Comms. 7:12062-8.