Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings.

Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodynamic and kinetic characterization at the single-molecule level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the electrical recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by molecules that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymatic reactions affected by product inhibition or when the release of products is the rate-limiting step.

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.

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore.

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.

Single-Molecule Analyte Recognition with ClyA Nanopores Equipped with Internal Protein Adaptors.

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.

A nanopore machine promotes the vectorial transport of DNA across membranes.

The transport of nucleic acids through membrane pores is a fundamental biological process that occurs in all living organisms. It occurs, for example, during the import of viral DNA into the host cell or during the nuclear pore complex-mediated transport of mRNA in and out the cell nucleus and has implications in nucleic acid drug delivery and gene therapy. Here we describe an engineered DNA transporter that is able to recognize and chaperone a specific DNA molecule across a biological membrane under a fixed transmembrane potential. The transported DNA strand is then released by a simple mechanism based on DNA strand displacement. This nanopore machine might be used to separate or concentrate nucleic acids or to transport genetic information across biological membranes.

Tuning the size and properties of ClyA nanopores assisted by directed evolution.

Nanopores have recently emerged as powerful tools in single-molecule investigations. Biological nanopores, however, have drawbacks, including a fixed size and limited stability in lipid bilayers. Inspired by the great success of directed evolution approaches in tailoring enzyme properties, in this work we evolved Cytolysin A from Salmonella typhi (ClyA) to a high level of soluble expression and desired electrical properties in lipid bilayers. Evolved ClyA nanopores remained open up to -150 mV applied potential, which allowed the detailed characterization of folded proteins by ionic current recordings. Remarkably, we also found that ClyA forms several nanopore species; among which we could isolate and characterize three nanopore types most likely corresponding to the 12mer, 13mer, and 14mer oligomeric forms of ClyA. Protein current blockades to the three ClyA nanopores showed that subnanometer variations in the diameter of nanopores greatly affect the recognition of analyte proteins.

An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.