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Unidirectional single-file transport of full-length proteins through a nanopore

Nature Biotechnology (2023)Cite this article

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Abstract

The electrical current blockade of a peptide or protein threading through a nanopore can be used as a fingerprint of the molecule in biosensor applications. However, threading of full-length proteins has only been achieved using enzymatic unfolding and translocation. Here we describe an enzyme-free approach for unidirectional, slow transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore, α-hemolysin (narrowest part is ~1.4 nm in diameter), and a high concentration guanidinium chloride buffer enables unidirectional, single-file protein transport propelled by an electroosmotic effect. We show that the mean protein translocation velocity depends linearly on the applied voltage and translocation times depend linearly on length, resembling the translocation dynamics of ssDNA. Using a supervised machine-learning classifier, we demonstrate that single-translocation events contain sufficient information to distinguish their threading orientation and identity with accuracies larger than 90%. Capture rates of protein are increased substantially when either a genetically encoded charged peptide tail or a DNA tag is added to a protein.

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Fig. 1: Enzyme-free full-length protein translocation through nanopores.
Fig. 2: Transport properties of unfolded protein analytes.
Fig. 3: MD simulation of ion, water and peptide transport through α-hemolysin.
Fig. 4: Unidirectional translocation and discrimination of MBP variants.
Fig. 5: Single-molecule fingerprinting of full-length MBP-D10 and GFP-D10.

Data availability

All data used in this manuscript are available for download at https://figshare.com/s/5cd39ee415c62a316a6f.

Code availability

All data parsing (excluding DTW and GBC) were performed using the Pyth-ion package (https://github.com/wanunulab/Pyth-Ion) and figures were generated using Igor. For analysis, the raw 100 kHz nanopore current data was further low-pass filtered to 10 kHz using the low-pass filter function in Pyth-ion. DTW and GBC analyses were conducted via python scripts written and documented in Jupyter Notebooks, tslearn (v0.5.2)77, SciKit-Learn (v1.0.2)78, and a modified version of the PyPore79 nanopore data analysis library. The Jupyter notebook and associated files are available on GitHub (https://github.com/wanunulab/protein-gd). A detailed description of the DTW and GBC analyses is provided in Supplementary Figs. 26–33, Supplementary Tables 4–7 and Supplementary Notes 3 and 4.

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Acknowledgements

We thank C. McCormick for assistance with editing the manuscript for clarity, and N. Slavov for helpful discussions regarding protein sequencing. We acknowledge funding from the National Institutes of Health under grants HG0011087 (to M.W.) and GM115442 (to M.C.), and the National Science Foundation under grant PHY-1430124 (to A.A.). The supercomputer time was provided through the XSEDE allocation grant (MCA05S028) and the Leadership Resource Allocation MCB20012 on Frontera of the Texas Advanced Computing Center.

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Authors and Affiliations

  1. Department of Physics, Northeastern University, Boston, MA, USA

    Luning Yu & Meni Wanunu

  2. Department of Bioengineering, Northeastern University, Boston, MA, USA

    Xinqi Kang, Amr Makhamreh, Ali Fallahi & Meni Wanunu

  3. Department of Chemistry, University of Massachusetts at Amherst, Amherst, MA, USA

    Fanjun Li & Min Chen

  4. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Behzad Mehrafrooz & Aleksei Aksimentiev

  5. Molecular and Cellular Biology Program, University of Massachusetts at Amherst, Amherst, MA, USA

    Joshua C. Foster & Min Chen

  6. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Aleksei Aksimentiev

  7. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Aleksei Aksimentiev

  8. Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA

    Meni Wanunu

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Contributions

L.Y. and M.W. conceived the project and designed the experiments. L.Y. developed the experimental protocol, carried out the experiments and analyzed the protein translocation data. X.K. and L.Y. fabricated the SU-8 wedge-on-pillar chips used in the experiments. F.L. and M.C. prepared and purified the protein samples. B.M. and A.A. designed and conducted the MD simulations. A.M. and A.F. performed the data analysis described in ‘protein-specific current signals’ (SDTW and GBC) and wrote the respective sections of the manuscript. J.C.F. performed the DNA–protein conjugation experiments. L.Y., B.M., A.A. and M.W. wrote the first draft of the manuscript, and all authors commented and edited it.

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Correspondence to Meni Wanunu.

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Supplementary Figs. 1–33, Supplementary notes 1–4 and Supplementary Tables 1–7.

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Yu, L., Kang, X., Li, F. et al. Unidirectional single-file transport of full-length proteins through a nanopore. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01598-3

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