Unfoldase-mediated protein translocation through an α-hemolysin nanopore

Abstract

Using nanopores to sequence biopolymers was proposed more than a decade ago1. Recent advances in enzyme-based control of DNA translocation2 and in DNA nucleotide resolution using modified biological pores3 have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned1. Although proteins have been shown to move through nanopores4,5,6, 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.

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Figure 1: Experimental set-up.
Figure 2: Ionic current traces during ClpX-mediated protein translocation.
Figure 3: Ionic current state dwell times during translocation of model proteins through the nanopore.

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Acknowledgements

The authors thank Oxford Nanopore Technologies (Oxford, UK) for supplying α-HL heptamers, and A. Martin (UC Berkeley) for supplying ClpX-related expression plasmids and for helpful discussions on their use. R. Abu-Shumays, D. Bernick, K. Lieberman and H. Olsen commented on drafts of the manuscript. This work was supported by a UC startup grant to M.A., and by equipment purchased previously using National Human Genome Research Institute grant R01HG006321. The ClpX-ΔN3 BLR expression strain was obtained from A. Martin (UC Berkeley), as was a his-tagged ClpP expression strain.

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Contributions

J.N. conceived and designed the project, performed the protein engineering and production, conceived and performed experiments and co-wrote the manuscript. D.B.M. performed and conceived experiments, analyzed data and co-wrote the manuscript. M.A. co-wrote the manuscript, designed the nanopore platform and directed the project.

Corresponding author

Correspondence to Mark Akeson.

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Competing interests

M.A. is a consultant to Oxford Nanopore Technologies, Oxford, UK.

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Nivala, J., Marks, D. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat Biotechnol 31, 247–250 (2013). https://doi.org/10.1038/nbt.2503

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