Real-Time Nanopore-Based Recognition of Protein Translocation Success

Abstract

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two "selectivity tags"-regions of different but uniform charge density-at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.

Figure 1

Salt-concentration-gradient-enhanced observation of α-syn dynamics in a VDAC nanopore. (a) Experimental setup (not to scale). The “diblock-copolymer”-like structure of α-syn is represented in color and consists of two “selectivity tags” of differing charge density (light yellow and dark red) that differently modulate the electrical properties of the VDAC nanopore under a salt concentration gradient. (b) Current-voltage curves of the open pore high-conducting (dark green circles) and the two low-conducting substates when α-syn is inside the pore (yellow triangles and red inverted triangles). Selectivities were calculated from the reversal potentials (vertical arrows); 68% confidence intervals are smaller than the size of the data points. (c) Sample current record. As-recorded data are shown in light gray and software-filtered data in dark blue. The total event duration, τ, was defined as shown. (d) Identification of the substates by noting that the capture of the CT in the nanopore corresponds to the lower-conducting substate. (e and f) Details of a retraction (e) and a translocation (f) event. The smooth overlay curve depicts $P_{\text{C}}\left(t\right)$ using the scaled representation $i_{\text{C}}\left(V\right)+\left(1-P_{\text{C}}\left(t\right)\right)\left(i_{\text{N}}\left(V\right)-i_{\text{C}}\left(V\right)\right)$ and a color scale between $P_{\text{C}}\left(t\right)=0$ (light yellow) and 1 (dark red). To see this figure in color, go online.

Figure 2

Direct experimental observation of translocation probability. (a) Fraction of events in which the uncharged selectivity tag was observed at the end of the event. Red circles represent the observed translocation probability; blue diamonds are corrected for the limited temporal resolution of the instrumentation. Error bars represent 68% confidence intervals. The solid line is the prediction of the drift-diffusion stochastic model for this system, whereas the dashed lines show 95% confidence intervals for this prediction. The model was optimized to the composite histograms of total event duration including both translocation and retraction events, but not to the experimentally measured translocation probability curve. (b) Average event durations with the optimized model. The average event duration is maximal at a transmembrane voltage of 50 mV, which corresponds to a pulling force on the CT of ≈7.4 pN. To see this figure in color, go online.

Figure 3

Average properties of retraction and translocation events. The substate occupancy is defined to be 0 when the NT is in the pore and 1 when the CT is in the pore. (a) Average substate occupancy as a function of time from the beginning of the event. Solid blue and yellow curves were averaged over retraction and translocation events, respectively, observed at a 45 mV transmembrane voltage, showing that both types of events begin with the charged region in the pore. Dotted lines are averages over all events at other voltages. Deviations from unity are caused by limited temporal resolution of the measurement, and they increase as the capture process shortens at higher voltages. (b) Average substate occupancy as a function of time from the end of the event, averaged over all retraction (top) and translocation (bottom) events. Average occupancy is fit by an exponential function that yields the characteristic timescales of the retraction $\langle {\tau }_{\text{ret}}\rangle$ and translocation $\langle {\tau }_{\text{trans}}\rangle$ processes. To see this figure in color, go online.