Unfoldase-mediated protein translocation through an α-hemolysin nanopore

Abstract

Using nanopores to sequence biopolymers was proposed more than a decade ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using modified biological pores have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned. Although proteins have been shown to move through nanopores, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore--a feature required for protein sequence analysis using this single-molecule technology.

Figure 1

(a) Nanopore sensor. A single α-HL pore is embedded in a lipid bilayer separating two polytetrafluoroethylene wells each containing 100 μl of 0.2 M KCl solution at 30 °C. Voltage is applied between the wells (trans side +180 mV), causing ionic current flow through the channel. Current diminishes in the presence of a captured protein molecule. (b) Protein capture in the nanopore. A model protein bearing an Smt3 domain (green) at its N terminus is coupled to a charged flexible linker (yellow) with an ssrA tag (red) at its C terminus. As a result of the applied voltage, the charged, flexible tag is threaded through the pore into the trans-side solution until the folded Smt3 domain prevents complete translocation of the captured protein. ClpX present in the trans solution binds the C-terminal ssrA sequence. Fueled by ATP hydrolysis, ClpX translocates along the protein tail toward the channel, and subsequently catalyzes unfolding and translocation of the Smt3 domain through the pore. (c) Engineered proteins used in this study. S1, a protein bearing a single N-terminal Smt3-domain coupled to a 65-amino-acid-long charged flexible segment capped at its carboxy terminus with the 11 amino acid ClpX-targeting domain (ssrA tag) (i); S2-35, similar to S1 but appended at its N terminus by a 35-amino-acid linker and a second Smt3 domain (ii); S2-148, identical to S2-35 except for an extended 148-amino-acid linker between the Smt3 domains (iii). The linker lengths in this panel are not to scale.

Figure 2

(a) S1 translocation. Open channel current through the α-HL nanopore under standard conditions (~34 ± 2 pA, RMS noise 1.2 ± 0.1 pA) (i). Capture of the S1 substrate. Upon protein capture, the ionic current drops to ~14 pA (~0.7 pA RMS noise) (ii). ClpX-mediated ramping state. The ionic current decreases to ~10 pA and is characterized by one or more gradual amplitude transitions. This pattern is only observed in the presence of ClpX and ATP (trans compartment) (iii). Smt3 domain unfolding and translocation through the nanopore (~3.8 pA, 1.7 pA RMS noise) (iv). Return to open channel current upon completion of substrate translocation to the trans compartment (i′). (b) Working model of ClpX-mediated translocation of S1. Roman numerals used to label panels correspond to ionic current states in a. (c) S2-35 translocation. Open channel current (i) is not shown. States ii–iv are identical to states ii–iv in a. Gradual increase in ionic current to ~10 pA. In our working model this corresponds to a transition from Smt3 domain translocation to linker region translocation (v). A second putative ramping state that closes resembles ramping state iii (vi). A second putative Smt3 translocation state with ionic current properties that closely resemble state iv (vii). Return to open channel current (i′). (d) S2-148 translocation. Ionic current states i–iv and vi–i′ were nearly identical to those states for S2-35 translocation in c. (v) In our working model, this ionic current state corresponds to translocation of the 148-amino-acid linker. Its amplitude is ~3 pA higher than the S2-35 linker amplitude (~9 pA), and it has a median duration ~2.5 fold longer than the comparable S2-35 state v. Translocation events that included ramping state iii were observed 62 times for protein S2-35 (7.3 h of experimentation), and 66 times for protein S2-148 (4.3 h of experimentation), when ClpX and ATP were present. In the absence of ClpX, these ramping states were never observed for S2-35 (1.7 h of experimentation) and S2-148 (1.2 h of experimentation).

Figure 3

(a) Comparison of putative Smt3 translocation (state iv) dwell times for three model proteins. Values are from events that included the ClpX-dependent ramping state (Fig. 2,iii). Black bars: median = 0.71 s, interquartile range (IQR) = 0.41, n = 45. Gray bars: median = 0.64 s, IQR = 0.47, n = 60. White bars: median = 0.63 s, IQR = 0.40, n = 65. (b) Comparison of putative linker region (state v) dwell times for S2-35 and S2-148 proteins. Values are from events that included the ClpX-dependent ramping state (Fig. 2,iii). Black bars: median = 1.52 s, IQR = 0.68, n = 50. Gray bars: median = 3.62 s, IQR = 2.03, n = 50. (c) State v translocation dwell times for S2-35 events. Black bars: median = 1.52 s, IQR = 0.68, n = 50. Gray bars: median = 11.45 s, IQR = 39.53, n = 45. (d) State v translocation dwell times for S2-148 translocation events. Black bars: median = 3.62 s, IQR = 2.03, n = 50. Gray bars: median = 37.07 s, IQR = 89.80, n = 20. (e) State ii dwell times for protein substrates at two voltages. Black bars: median = 4.89 s, IQR = 6.72, n = 104. White bars: median = 165.50 s, IQR = 351.75, n = 34. Gray bars: median = 17.26 s, IQR = 31.08, n = 173. Light gray bars: median = 90.0 s, IQR = 112.0, n = 72. (f) State iv dwell times for the S1 protein substrate at two voltages. Black bars: median = 0.33 s, IQR = 0.40, n = 104. White bars: median = 8.25 s, IQR = 26.82, n = 34. Gray bars: median = 0.65 s, IQR = 0.45, n = 52. Light gray bars: median = 1.74 s, IQR = 3.12, n = 41.