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

Nature Chemistry volume 13, pages 1192–1199 (2021)Cite this article

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Abstract

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.

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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.

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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.

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Acknowledgements

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

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Authors and Affiliations

  1. Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands

    Shengli Zhang, Gang Huang, Roderick Corstiaan Abraham Versloot, Bart Marlon Herwig Bruininks, Paulo Cesar Telles de Souza, Siewert-Jan Marrink & Giovanni Maglia

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Contributions

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.

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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.

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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). https://doi.org/10.1038/s41557-021-00824-w

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