Single-molecule nanopore sensing of actin dynamics and drug binding

Abstract

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.through different modes of action. Finally, we demonstrate the ability of nanopipette-based nanopores in drug discovery by measuring real-time drug-binding at a single molecule level, calculating the observed rate constant and the saturation concentration of Swinholide A dimer formation. Using these measurements, we propose a positively cooperative actin-binding model for the Swinholide A drug's mode of action. The ability to perform such studies, label-free and with single-molecule resolution demonstrates the potential of using quartz nanopipettes in both the direct probing of the unfolding of complex biomolecules but also for future molecular diagnostics and drug discovery.6 Results Experimental configurationNanopipettes were fabricated using laser-assisted pulling as reported previously 28 . The pulled nanopipettes terminated with a single nanopore with an average diameter of 25 ± 4 nm (N = 5), as measured by Scanning Electron Microscopy (Fig. 1a and Fig. S1). Linear IV curves were obtained in the monomeric actin buffer, and the nanopore conductance was determined to be 37.0 ± 3.9 nS (Fig. S2). Given the high potassium chloride concentration (1 M), we confirmed that actin remained monomeric in the nanopore buffer (Fig. S3). In all experiments, the analyte was introduced into an external reservoir (CIS) along with a reference/ground Ag/AgCl electrode. The pipette was filled only with buffer solution, and a patch/working Ag/AgCl electrode was inserted ( Fig. 1a). When an external electric field is applied, protein transport through the nanopore is a result of cooperative and competitive contributions from diffusion, electrophoretic (EP) and electroosmotic (EO) flows 29 . At pH 8.0, both the surface of the nanopipettes and actin (isoelectric point ~5.3) are negatively charged, meaning we see no translocation events at a negative voltage (Fig. S4). We used rel...

Modulation of electrophoresis, electroosmosis and diffusion for electrical transport of proteins through a solid-state nanopore

“…3,41 An unfolded protein is typically more surface charged than its folded counterpart due to the exposure of charged moieties that are otherwise hidden due to folding. 42 According to the Hofmeister series, Li + is a chaotropic cation while K + is a kosmotropic cation. 43 It is possible that with voltage-driven unfolding, the ions of the electrolyte would have more access to the otherwise shielded moieties due to the folded structure with chaotropic cations denaturing and destabilizing the protein structure compared to kosmotropic cations.…”

Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum

“…All the nanopipettes were fabricated by pulling quartz capillaries (GQF100-50-7.5, World Precision Instruments, UK) using a laser-assisted pipette puller (Sutter Instrument, P-2000, USA) as per protocol reported previously by our group 33 , 62 . Prior to pulling, the capillaries (inner diameter: 0.5 mm, outer diameter: 1.0 mm, length: 7.5 cm) were cleaned thoroughly for approximately 30 min using a plasma cleaner (Harrick Plasma) to remove any organic residues or contaminants on the quartz surface.…”

Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore

“… 45 The formation of amyloid fibrils and other filamentous proteins has been studied with nanopores, but complex surface modifications of the nanopore are often required. 46 − 49 Having demonstrated that macromolecular crowding improves the nanopore sensitivity for globular proteins, we then investigated whether the detection of α-synuclein amyloid fibrils could also be enhanced and whether the length of the amyloid fibrils affected the translocation dynamics. In order to analyze fibrils with distinct length distributions, fragmented and elongated α-synuclein amyloid fibrils were generated (see the Methods in the Supporting Information ).…”