Engineering and Modeling the Electrophoretic Trapping of a Single Protein Inside a Nanopore

Kherim Willems, Dino Ruić, Annemie Biesemans, Nicole Stéphanie Galenkamp, Pol Van Dorpe, Giovanni Maglia*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here we describe a system where steric, electrostatic, electrophoretic, and electroosmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed retrieving direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electroosmotic flow on the protein (≅9 pN at -50 mV). The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies.

Original languageEnglish
Pages (from-to)9980-9992
Number of pages13
JournalAcs Nano
Issue number9
Early online date12-Aug-2019
Publication statusPublished - 12-Aug-2019


  • ClyA nanopore
  • DHFR
  • electrostatic trap
  • electro-osmotic flow
  • protein electrostatics
  • nanomanipulation
  • single-molecule enzymology
  • DNA

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