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Unfoldase-mediated protein translocation through an α-hemolysin nanopore

Nature Biotechnology volume 31, pages 247250 (2013) | Download Citation


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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  1. 1.

    , , , & Characterization of individual polymer molecules based on monomer-interface inter- action. US patent 5,795,782 (1998).

  2. 2.

    et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat. Biotechnol. 30, 344–348 (2012).

  3. 3.

    et al. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat. Biotechnol. 30, 349–353 (2012).

  4. 4.

    , , & Controlling a single protein in a nanopore through electrostatic traps. J. Am. Chem. Soc. 130, 4081–4088 (2008).

  5. 5.

    & Single-molecule protein unfolding in solid state nanopores. J. Am. Chem. Soc. 131, 9287–9297 (2009).

  6. 6.

    et al. Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording. ACS Chem. Biol. 7, 652–658 (2012).

  7. 7.

    & ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochim. Biophys. Acta 1823, 15–28 (2012).

  8. 8.

    et al. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145, 257–267 (2011).

  9. 9.

    et al. ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145, 459–469 (2011).

  10. 10.

    & A protein-conducting channel in the endoplasmic reticulum. Cell 65, 371–380 (1991).

  11. 11.

    , , & The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16, 5509–5519 (1997).

  12. 12.

    & Solution structure of a yeast ubiquitin-like protein Smt3: the role of structurally less defined sequences in protein-protein recognitions. Protein Sci. 11, 1482–1491 (2002).

  13. 13.

    , , & The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338–1347 (1998).

  14. 14.

    et al. Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. Mol. Cell 5, 639–648 (2000).

  15. 15.

    et al. Effect of charge, topology and orientation of the electric field on the interaction of peptides with the α-hemolysin pore. J. Pept. Sci. 17, 726–734 (2011).

  16. 16.

    Interrogating single proteins through nanopores: challenges and opportunities. Trends Biotechnol. 27, 333–341 (2009).

  17. 17.

    et al. Unfolding of proteins and long transient conformations detected by single nanopore recording. Phys. Rev. Lett. 98, 158101 (2007).

  18. 18.

    et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nat. Nanotechnol. 5, 798–806 (2010).

  19. 19.

    , & Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 (2005).

  20. 20.

    , , & Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine. Cell 114, 511–520 (2003).

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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.

Author information


  1. Nanopore Group, Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, California, USA.

    • Jeff Nivala
    • , Douglas B Marks
    •  & Mark Akeson


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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.

Competing interests

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

Corresponding author

Correspondence to Mark Akeson.

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