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Real-time shape approximation and fingerprinting of single proteins using a nanopore

Nature Nanotechnology volume 12, pages 360–367 (2017)Cite this article

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Abstract

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

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Figure 1: Rotational dynamics of individual proteins inside a nanopore reveal a spheroidal approximation of the protein's shape m.
Figure 2: Three different strategies of anchoring proteins to the lipid coating used in this work to slow down translocation such that rotational diffusion of the proteins could be resolved in time.
Figure 3: Determination of approximate protein shape and volume from histograms of maximum ΔI values from resistive pulse recordings.
Figure 4: Approximate shape, dipole moment and rotational diffusion coefficient obtained from current modulations within individual resistive pulses from the translocation of a single protein.
Figure 5: Fingerprinting of individual translocation events permits identification and characterization of G6PDH and a G6PDH–IgG complex from a binary mixture.

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Acknowledgements

We thank C. L. Asbury for several helpful discussions and review of the manuscript. This work was supported by a Miller Faculty Scholar Award (M.M.), the Air Force Office of Scientific Research (M.M. and D.S., grant number FA9550-12-1-0435), Oxford Nanopore Technologies (M.M., grant number 350509-N016133), the National Institutes of Health (M.M., grant number 1R01GM081705), the National Human Genome Research Institute (J.L., grant numbers HG003290 and HG004776), J. Golovchenko's Harvard nanopore group for FIB pore preparation (J.L.), a Rackham Pre-Doctoral Fellowship from the University of Michigan (E.C.Y.), a Graduate Research Fellowship from the National Science Foundation (J.H.) and the Microfluidics in Biomedical Sciences Training Program from the NIH and BIBIB (B.R.B.).

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Author notes

  1. Erik C. Yusko and Brandon R. Bruhn: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Erik C. Yusko, Brandon R. Bruhn, Olivia M. Eggenberger, Jared Houghtaling, David Sept & Michael Mayer

  2. Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, Fribourg, CH-1700, Switzerland

    Olivia M. Eggenberger, Jared Houghtaling & Michael Mayer

  3. Department of Physics, University of Arkansas, Fayetteville, 72701, Arkansas, USA

    Ryan C. Rollings, Nathan C. Walsh, Santoshi Nandivada & Jiali Li

  4. Department of Pharmaceutical Sciences, University of Connecticut, Storrs, 06269, Connecticut, USA

    Mariya Pindrus & Devendra S. Kalonia

  5. Department of Biomedical Engineering and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston Salem, 27157, North Carolina, USA

    Adam R. Hall

  6. Center for Computational Medicine and Biology, University of Michigan, Ann Arbor, 48109, Michigan, USA

    David Sept

  7. Biophysics Program, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Michael Mayer

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Contributions

E.C.Y., B.R.B. and M.M. conceived and designed experiments, analysed data, and co-wrote the manuscript. E.C.Y., B.R.B., J.H. and O.M.E. performed nanopore experiments. R.C.R., N.C.W., S.N., A.R.H. and J.L. fabricated nanopores. M.P, D.S.K. and B.R.B. measured the dipole moments of proteins with impedance spectroscopy and provided constructive feedback on the manuscript. D.S. performed computational analyses of several protein crystal structures and provided guidance on statistical methods used in the manuscript.

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Correspondence to Michael Mayer.

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Yusko, E., Bruhn, B., Eggenberger, O. et al. Real-time shape approximation and fingerprinting of single proteins using a nanopore. Nature Nanotech 12, 360–367 (2017). https://doi.org/10.1038/nnano.2016.267

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