Abstract 1356 Modulating Flow through a Protein Nanopore

The ability to control the flow of molecular species through a nanopore impacts many applications of nanoscience such as delivery of molecular cargo, drug targeting, and nanoscale manufacturing. We seek to develop and characterize mechanisms that allow for controllable, reversible, and repetitive opening and closing of protein nanopores. The self-assembling exotoxin alpha-hemolysin (⍺HL) is responsible for the lytic activity during Staphylococcus aureus infection and serves as the prototype for switchable nanopores in our study. In this work, we derivatize ⍺HL with polypeptide histidine (His6) tags and examine the ability of these modified nanopores to respond to Ni2+ ions. Our findings using two distinct assay methods indicate attenuation of flow through His6-⍺HL nanopores in the presence of Ni2+. Significantly, lysis of red blood cells by His6-⍺HL nanopores can be arrested by the addition of Ni2+. Additionally, electrophysiological interrogation of single His6-⍺HL nanopores shows drastically reduced channel conductance upon addition of Ni2+. Both assay methods allow measurement of reversible and repetitive nanopore opening (in the absence of Ni2+) and closing (in the presence of Ni2+). Importantly, this switching behavior is unique to the His6-derivatized ⍺HL over wildtype, suggesting that the His6 tags on the heptameric nanopore bind Ni2+ and obstruct flow through the pore. We present data comparing wildtype and His6 versions of ⍺HL including cell lysis curves, phase contrast images, Ni2+ titrations, current conductance traces, and reversibility studies. Our results describe a His6/Ni2+ actuated nanopore system that is amenable to fine-tuned modulation. Support for this work was provided by the National Science Foundation (CHE Grant #2105892) and the Wheaton College Alumni Association.