SDS-assisted protein transport through solid-state nanopores

Abstract

Using nanopores for single-molecule sequencing of proteins - similar to nanopore-based sequencing of DNA - faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores.

Figure 1

Schematic illustration and representative current traces of the translocation of native protein (a) and SDS-unfolded protein (b) through a solid-state nanopore. The traces correspond to the translocation of β−amylase through a 10 nm nanopore.

Figure 2

Microscopic models of SDS/protein complexes. (a) MD simulation of titin/SDS complex self-assembly. The sequence of snapshots illustrates the microscopic state of the system during a 300 ns explicit solvent all-atom MD simulation. The protein backbone is shown in blue, SDS molecules in cyan and red, water and 0.4 M NaCl are now shown. (b) Typical microscopic conformations of the SDS/protein assemblies obtained at the end of the self-assembly simulations. The SDS molecules are shown using the molecular bonds representation: Carbon, sulfur and oxygen atoms are shown in cyan, yellow and red, respectively; hydrogen atoms are not shown. Each protein molecule is shown as a trace of the protein backbone.

Figure 3

MD simulation of SDS/protein nanopore translocation. (a-d) Simulated ionic current blockades produced by translocation of various molecular species through a 6 nm diameter nanopore (System 1). Left column illustrates typical microscopic conformations observed during nanopore translocation simulations; the black arrow indicated the direction of the positive transmembrane bias. Protein conformation is depicted as a trace of the protein backbone; SDS molecules are shown as molecular bonds; water and ions are not shown for clarity. Right columns shows the ionic current traces recorded from the translocation simulations and the open pore current level. Because of the periodic boundary conditions employed in our MD simulations, individual ionic current traces feature multiple translocation events. Ionic current traces from independent simulations are delineated by the “//” mark. Black circles and horizontal black bars indicate the average blockade current of individual blockade events and the duration of each event, respectively. With the exception of native titin, the blockade events were defined by the reduction of the nanopore current below 75% of the open pore value; the blockade event in the native titin simulation was characterized using the titin’s center of mass coordinates. (e) Sequence of snapshots illustrating a nanopore translocation of a β-amylase/SDS complex. (f) Same as in panels a-d but for an SDS micelle. (g) Average translocation time of the SDS/protein assemblies. The color of the bars indicates the transmembrane voltage. (h) The average conductance blockade amplitudes. To enable direct comparison with experiment, the conductance blockades computed from MD simulations were scaled by the ratio of the experimental and simulation bulk conductivity of 0.4 KCl (3.6/4.8). Open bars indicate conductance blockade obtained from the molecular assemblies placed at the center of a 10 nm diameter nanopore (System 2) at a 500 mV bias; the molecular configurations are defined in SI Figure S3.

Figure 4

(a) Schematic of the dominant mechanisms of protein translocation at SDS concentrations below and above the CMC. (b) Event rate of the translocation of titin-SDS complexes through a 10 nm solid-state nanopore at different SDS concentrations. At SDS concentrations below the CMC (blue) electro-osmosis is the dominant transport mechanism of titin-SDS through solid-state nanopores. At SDS concentrations above the CMC (green) electrophoresis dominates. (c) Plot of the event rate vs. SDS concentration for SDS micelles alone and SDS-unfolded proteins.

Figure 5

Translocation of SDS micelles through a 10 nm solid-state nanopore (a) Schematic representation of the nanopore control experiment. (b) Example of a current trace measured showing the translocation of SDS micelles. (c) Dwell time vs. conductance blockade scatter plot for SDS-micelles and SDS-treated protein. (d) Conductance blockade histogram of the translocation of micelles.

Figure 6

(a) Current trances of the translocation of native β−amylase and SDS-unfolded β−amylase through a 10 nm pore at 10 kHz bandwidth. Typical events are shown in the right panels. (b) Scatter plot of conductance blockade vs. dwell time of native β−amylase and SDS-treated β−amylase. Native protein translocations are measured in the direction of the electro-osmotic flow, SDS-treated proteins are measured in the electrophoretic direction. (c) Histograms of conductance blockade of native protein (left), SDS-treated protein bellow CMC (middle), and SDS-treated protein above CMC (right). The conductance blockade of the native protein has a value of 3.7 ± 1.7 nS. The blockade for the SDS-treated protein below CMC is 0.53 ± 0.29 nS. Above CMC, two peaks are observed. The first has a value of 0.40 ± 0.27 nS, and the second one, presumably due to SDS micelles, has a value of 4.3 ± 0.4 nS.