Abstract
Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap’s ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.
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Data availability
Data are available at https://doi.org/10.5281/zenodo.5059802.
Code availability
Code for data analysis of nanopore recordings as described herein are available at https://doi.org/10.5281/zenodo.5059802.
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Acknowledgements
Hsp90 was a gift from B. Hermann and T. Hugel. Avidin was a gift from M. Howarth. ClpP and ClpX plasmids were a gift from C. Joo. We thank X. Shi and A. Fragasso for discussions, E. van der Sluis for discussions and protein purification, M.-Y. Wu and F. Tichelaar for TEM drilling. The work was funded by NWO-I680 (SMPS) and supported by the NWO/OCW Gravitation program NanoFront and the European Research Council Advanced grant no. 883684. S.S. acknowledges the Postdoc.Mobility fellowship no. P400PB_180889 by the Swiss National Science Foundation. This work was supported by a European Research Council Consolidator grant to H.D. (grant agreement no. 724261), the Deutsche Forschungsgemeinschaft through grants provided within the Gottfried-Wilhelm-Leibniz Program (to H.D.).
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S.S. and C.D. conceived the project. S.S. performed all nanopore experiments, analysed the data and purified proteins. H.D. and P.S. advised on DNA origami, and P.S. folded and characterized it. S.S. wrote the paper with C.D. All authors discussed the results and commented on the paper.
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Supplementary Figs 1–9, Table 1, Notes 1–4 and References.
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Schmid, S., Stömmer, P., Dietz, H. et al. Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations. Nat. Nanotechnol. 16, 1244–1250 (2021). https://doi.org/10.1038/s41565-021-00958-5
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DOI: https://doi.org/10.1038/s41565-021-00958-5
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