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

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 electro-osmotic 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 direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electro-osmotic flow on the protein (≅9 pN at -50 mV) to be retrieved. 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.

Jaar van publicatie:2019

BOF-keylabel:ja

IOF-keylabel:ja

BOF-publication weight:10

CSS-citation score:2

Auteurs:International

Authors from:Government, Higher Education

Toegankelijkheid:Open