9 research outputs found
Single-Molecule Sampling of Dihydrofolate Reductase Shows Kinetic Pauses and an Endosteric Effect Linked to Catalysis
[Image: see text] The ability to sample multiple reactions on the same single enzyme is important to link rare intermediates with catalysis and to unravel the role of conformational changes. Despite decades of efforts, however, the single-molecule characterization of nonfluorogenic enzymes during multiple catalytic turnovers has been elusive. Here, we show that nanopore currents allow sampling the dynamic exchange between five structural intermediates during E. coli dihydrofolate reductase (DHFR) catalysis. We found that an endosteric effect promotes the binding of the substrate to the enzyme with a specific hierarchy. The chemical step then switched the enzyme from the closed to the occluded conformation, which in turn promotes the release of the reduced cofactor NADP(+). Unexpectedly, only a few reactive complexes lead to catalysis. Furthermore, second-long catalytic pauses were observed, possibly reflecting an off-path conformation generated during the reaction. Finally, the free energy from multiple cofactor binding events were required to release the product and switch DHFR back to the reactive conformer. This catalytic fueled concerted mechanism is likely to have evolved to improve the catalytic efficiency of DHFR under the high concentrations of NADP(+) in E. coli and might be a general feature for complex enzymatic reactions where the binding and release of the products must be tightly controlled
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 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
Current Blockades of Proteins Inside Nanopores for Real-Time Metabolome Analysis
Biological nanopores are emerging as powerful and low-cost sensors for real-time analysis of biological samples. Proteins can be incorporated inside the nanopore, and ligand binding to the protein adaptor yields changes in nanopore conductance. In order to understand the origin of these conductance changes and develop sensors for detecting metabolites, we tested the signal originating from 13 different protein adaptors. We found that the quality of the protein signal depended on both the size and charge of the protein. The engineering of a dipole within the surface of the adaptor reduced the current noise by slowing the protein dynamics within the nanopore. Further, the charge of the ligand and the induced conformational changes of the adaptor defined the conductance changes upon metabolite binding, suggesting that the protein resides in an electrokinetic minimum within the nanopore, the position of which is altered by the ligand. These results represent an important step toward understanding the dynamics of the electrophoretic trapping of proteins inside nanopores and will allow developing next-generation sensors for metabolome analysis.status: publishe
Direct electrical quantification of glucose and asparagine from bodily fluids using nanopores
Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics
Automated Electrical Quantification of Vitamin B1 in a Bodily Fluid using an Engineered Nanopore-Sensor
The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore‐based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the π‐π stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in a marked difference between unbound and bound state, allowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only. Using an array of nanopores, this will allow the quantification within seconds, paving the way for next‐generation single‐molecule metabolite detection systems
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. [Figure not available: see fulltext.]
Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor
Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers
Single-Molecule Kinetics of Growth and Degradation of Cell-Penetrating Poly(disulfide)s
The delivery of therapeutic agents into target cells is a challenging task. Cell penetration and intracellular targeting were recently addressed with biodegradable cell-penetrating poly(disulfide)s (CPDs). Cellular localization is determined by the length of these polymers, emphasizing the significance of initial chain length and the kinetics of intracellular depolymerization for targeted delivery. In the present study, the kinetics of CPD polymer growth and degradation were monitored in a single-molecule nanoreactor. The chain lengths achievable under synthetic conditions with high concentrations of dithiolanes were then predicted by using the rate constants. For example, CPDs comprising 40 units are generated in 1 s at pH 7.4 and 0.3 s at pH 8.4 at dithiolane concentrations of 200 mM. The rate constants for degradation suggest that the main depolymerization pathway in the cell is by monomer removal by self-cyclization, rather than by intrachain cleavage by endogenous thiols
Preparation of Fragaceatoxin C (FraC) Nanopores
Biological nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) nanopores, which may have applications for DNA analysis and nanopore-based proteomics. Recombinantly expressed FraC nanopores are purified via two rounds of Ni-NTA affinity chromatography before and after oligomerization on sphingomyelin-containing liposomes. Starting from a plasmid vector containing the FraC gene, our method allows the production of purified nanopores within a week. Afterward, the FraC nanopores can be stored at +4 °C for several months, or frozen