The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.Keywords: nanopore; nanotechnology; protein modifications; single-molecule kinetics; ubiquitin;

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.Keywords: barrier; dsDNA; mechanism; ssDNA; translocation; transport

Rotaxanes, pseudorotaxanes, and catenanes are supramolecular complexes with potential use in nanomachinery, molecular computing, and single-molecule studies. Here we constructed a protein rotaxane in which a polypeptide thread is encircled by a Cytolysin A (ClyA) nanopore and capped by two protein stoppers. The rotaxane could be switched between two states. At low negative applied potentials (<−50 mV) one of the protein stoppers resided inside the nanopore indefinitely. Under this configuration the rotaxane prevents the diffusion of protein molecules across the lipid bilayer and provides a useful platform for single-molecule analysis. High negative applied potentials (−100 mV) dismantled the interlocked rotaxane system by the forceful translocation of the protein stopper, allowing new proteins to be trapped inside or transported across the nanopore. The observed voltage threshold for the translocation of the protein stopper through the nanopore related well to the biphasic voltage dependence of the residence time measured for the freely diffusing protein stopper. We propose a model in which molecules translocate through a nanopore when the average dwell time decreases with the applied potential.

Nanopores have been used to detect molecules, to sequence DNA, or to investigate chemical reactions at the single-molecule level. Because they approach the absolute limit of sensor miniaturization, nanopores are amenable to parallelization and could be used in single-cell measurements. Here we show that single enzymes can be functionally and reversibly trapped inside the confined space of a ClyA nanopore. Remarkably, the binding of ligands to the internalized proteins is mirrored by specific changes to the nanopore conductance. Conveniently, the manipulation of the charge of the protein allowed increasing of the residence time of the protein inside the nanopore. Nanopores with internalized protein adaptors can be used to study proteins in real time or can be incorporated into inexpensive portable devices for the detection of analytes with high selectivity.