TY - JOUR

T1 - Engineering and Modeling the Electrophoretic Trapping of a Single Protein Inside a Nanopore

AU - Willems, Kherim

AU - Ruić, Dino

AU - Biesemans, Annemie

AU - Galenkamp, Nicole Stéphanie

AU - Van Dorpe, Pol

AU - Maglia, Giovanni

PY - 2019/8/12

Y1 - 2019/8/12

N2 - 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.

AB - 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.

KW - ClyA nanopore

KW - DHFR

KW - electrostatic trap

KW - electro-osmotic flow

KW - protein electrostatics

KW - nanomanipulation

KW - single-molecule enzymology

KW - PEP-FOLD

KW - MOLECULES

KW - FORCE

KW - DNA

KW - TRANSLOCATION

KW - DYNAMICS

KW - OBJECTS

KW - ELECTROSTATICS

KW - NANOPARTICLES

KW - BIOMOLECULES

U2 - 10.1021/acsnano.8b09137

DO - 10.1021/acsnano.8b09137

M3 - Article

C2 - 31403770

VL - 13

SP - 9980

EP - 9992

JO - Acs Nano

JF - Acs Nano

SN - 1936-0851

IS - 9

ER -