Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Bottom-up fabrication of a proteasome–nanopore that unravels and processes single proteins


The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise β-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Design of a proteasome–nanopore.
Fig. 2: Fabrication and optimization of the artificial nanopores.
Fig. 3: Electrical properties of the REG–nanopore.
Fig. 4: Design of the artificial proteasome–nanopore.
Fig. 5: Controlled translocation through the proteasome–nanopore.

Data availability

All relevant data are included in the article and its Supplementary Information. Statistical source data, unmodified gels, and molecular dynamics simulations results are provided in Source data. Data is also available from the authors upon reasonable request. Source data are provided with this paper.


  1. 1.

    Bayley, H. Nanopore sequencing: from imagination to reality. Clin. Chem. 61, 25–31 (2014).

    Article  Google Scholar 

  2. 2.

    Kang, X. F., Cheley, S., Guan, X. & Bayley, H. Stochastic detection of enantiomers. J. Am. Chem. Soc. 128, 10684–10685 (2006).

    CAS  Article  Google Scholar 

  3. 3.

    Huang, G., Voet, A. & Maglia, G. FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution. Nat. Commun. 10, 1–10 (2019).

    Article  Google Scholar 

  4. 4.

    Huang, G., Willems, K., Soskine, M., Wloka, C. & Maglia, G. Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat. Commun. 8, 935 (2017).

  5. 5.

    Restrepo-Pérez, L., Wong, C. H., Maglia, G., Dekker, C. & Joo, C. Label-free detection of post-translational modifications with a nanopore. Nano Lett. 19, 7957–7964 (2019).

    Article  Google Scholar 

  6. 6.

    Ouldali, H. et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat. Biotechnol. 38, 176–181 (2020).

    CAS  Article  Google Scholar 

  7. 7.

    Hu, Z.-L., Huo, M.-Z., Ying, Y.-L. & Long, Y.-T. Biological nanopore approach for single‐molecule protein sequencing. Angew. Chemie 60, 14738–14749 (2020).

    Article  Google Scholar 

  8. 8.

    Nivala, J., Mulroney, L., Li, G., Schreiber, J. & Akeson, M. Discrimination among protein variants using an unfoldase-coupled nanopore. ACS Nano 8, 12365–12375 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Nivala, J., Marks, D. B. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 31, 247–250 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Xu, C. et al. Computational design of transmembrane pores. Nature 585, 129–134 (2020).

    CAS  Article  Google Scholar 

  11. 11.

    Joh, N. H. et al. De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 346, 1520–1520 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Lu, P. et al. Accurate computational design of multipass transmembrane proteins. Science 359, 1042–1046 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Scott, A. et al. Constructing ion channels from water-soluble α-helical barrels. Nat. Chem. 13, 643–650 (2021).

  14. 14.

    Spruijt, E., Tusk, S. E. & Bayley, H. DNA scaffolds support stable and uniform peptide nanopores. Nat. Nanotechnol. 13, 739–745 (2018).

    CAS  Article  Google Scholar 

  15. 15.

    Seemüller, E. et al. Proteasome from Thermoplasma acidophilum: a threonine protease. Science 268, 579–582 (1995).

    Article  Google Scholar 

  16. 16.

    Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533–539 (1995).

    Article  Google Scholar 

  17. 17.

    Sugiyama, M. et al. Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form. Biochem. Biophys. Res. Commun. 432, 141–145 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Förster, A., Masters, E. I., Whitby, F. G., Robinson, H. & Hill, C. P. The 1.9 Å structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589–599 (2005).

    Article  Google Scholar 

  19. 19.

    Jiang, J., Pentelute, B. L., Collier, R. J. & Hong Zhou, Z. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521, 545–549 (2015).

    CAS  Article  Google Scholar 

  20. 20.

    Cheley, S., Braha, O., Lu, X., Conlan, S. & Bayley, H. A functional protein pore with a "retro" transmembrane domain. Protein Sci. 8, 1257–1267 (1999).

    CAS  Article  Google Scholar 

  21. 21.

    Gu, L. Q. et al. Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters. Proc. Natl Acad. Sci. USA 97, 3959–3964 (2000).

    CAS  Article  Google Scholar 

  22. 22.

    Maglia, G., Restrepo, M. R., Mikhailova, E. & Bayley, H. Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge. Proc. Natl Acad. Sci. USA 105, 19720–19725 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    Chen, B. et al. Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase. Structure 18, 1420–1430 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    Gu, L. Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690 (1999).

    CAS  Article  Google Scholar 

  25. 25.

    Yannakopoulou, K. et al. Symmetry requirements for effective blocking of pore-forming toxins: comparative study with α-, β-, and γ-cyclodextrin derivatives. Antimicrob. Agents Chemother. 55, 3594–3597 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Förster, A. & Hill, C. P. Proteasome activators. Protein Degrad. 2, 89–110 (2007).

    Article  Google Scholar 

  27. 27.

    Huber, E. M. & Groll, M. The mammalian proteasome activator PA28 forms an asymmetric α4β3 complex. Structure 25, 1473–1480.e3 (2017).

    CAS  Article  Google Scholar 

  28. 28.

    Kuehn, L. & Dahlmann, B. Proteasome activator PA28 and its interaction with 20S proteasomes. Arch. Biochem. Biophys. 329, 87–96 (1996).

    CAS  Article  Google Scholar 

  29. 29.

    Benaroudj, N., Zwickl, P., Seemüller, E., Baumeister, W. & Goldberg, A. L. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69–78 (2003).

    CAS  Article  Google Scholar 

  30. 30.

    Huang, R. et al. Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study. Proc. Natl Acad. Sci. USA 113, E4090–W4199 (2016).

    Google Scholar 

  31. 31.

    Ripstein, Z. A., Huang, R., Augustyniak, R., Kay, L. E. & Rubinstein, J. L. Structure of a AAA+ unfoldase in the process of unfolding substrate. eLife 6, 1–14 (2017).

    Article  Google Scholar 

  32. 32.

    Gerega, A. et al. VAT, the Thermoplasma homolog of mammalian p97/VCP, is an N domain-regulated protein unfoldase. J. Biol. Chem. 280, 42856–42862 (2005).

    CAS  Article  Google Scholar 

  33. 33.

    Akopian, T. N., Kisselev, A. F. & Goldberg, A. L. Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. J. Biol. Chem. 272, 1791–1798 (1997).

    CAS  Article  Google Scholar 

  34. 34.

    Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  Google Scholar 

  35. 35.

    Ward, W. W., Prentice, H. J., Roth, A. F., Cody, C. W. & Reeves, S. C. Spectral perturbations of the aequorea green-fluorescent protein. Photochem. Photobiol. 35, 803–808 (1982).

    CAS  Article  Google Scholar 

  36. 36.

    Hsu, S.-T. D., Blaser, G. & Jackson, S. E. The folding, stability and conformational dynamics of β-barrel fluorescent proteins. Chem. Soc. Rev. 38, 2951–2965 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Kisselev, A. F., Songyang, Z. & Goldberg, A. L. Why does threonine, and not serine, function as the active site nucleophile in proteasomes? J. Biol. Chem. 275, 14831–14837 (2000).

    CAS  Article  Google Scholar 

  38. 38.

    Biesemans, A., Soskine, M. & Maglia, G. A protein rotaxane controls the translocation of proteins across a ClyA nanopore. Nano Lett. 15, 6076–6081 (2015).

    CAS  Article  Google Scholar 

  39. 39.

    Majumder, P. et al. Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle. Proc. Natl Acad. Sci. USA 116, 534–539 (2019).

    CAS  Article  Google Scholar 

  40. 40.

    Kisselev, A. F., Akopian, T. N. & Goldberg, A. L. Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes. J. Biol. Chem. 273, 1982–1989 (1998).

    CAS  Article  Google Scholar 

  41. 41.

    Maglia, G., Heron, A. J. J., Stoddart, D., Japrung, D. & Bayley, H. Analysis of single nucleic acid molecules with protein nanopores. Methods Enzym. 475, 591–623 (2010).

    CAS  Article  Google Scholar 

Download references


This work is financially supported by ERC consolidator grant (no. 726151).

Author information




S.Z. and G.M. designed the experiments. G.M. supervised the project. S.Z. performed the experiments and data analysis. B.M.H.B., P.C.T.d.S. and S.-J.M. conducted the simulation work. G.M. and S.Z. wrote the paper. All authors discussed the results, and commented on the manuscript.

Corresponding author

Correspondence to Giovanni Maglia.

Ethics declarations

Competing interests

G.M. is a founder, director and shareholder of Portal Biotech Limited, a company engaged in the development of nanopore technologies. This work was not supported by Portal Biotech Limited.

Additional information

Peer review information Nature Chemistry thanks Ulrich Keyser, Yi-Lun Ying and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Additional discussion, methods, Supplementary Figs. 1–29, Table 1 and references.

Supplementary Data 1

molecular dynamics simulations of REG–nanopore for Fig. 1d.

Supplementary Data 2

molecular dynamics simulations of proteasome–nanopore for Fig. 1h.

Supplementary Data 3

Statistical Source Data for Supplementary Fig. 7.

Supplementary Data 4

Statistical Source Data for Supplementary Fig. 28.

Supplementary Data

Unprocessed gels.

Source data

Source Data Fig. 3

Statistical Source Data.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 5

Statistical Source Data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, S., Huang, G., Versloot, R.C.A. et al. Bottom-up fabrication of a proteasome–nanopore that unravels and processes single proteins. Nat. Chem. 13, 1192–1199 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing