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.

Real-time measurement of protein–protein interactions at single-molecule resolution using a biological nanopore


Protein–protein interactions (PPIs) are essential for many cellular processes. However, transient PPIs are difficult to measure at high throughput or in complex biological fluids using existing methods. We engineered a genetically encoded sensor for real-time sampling of transient PPIs at single-molecule resolution. Our sensor comprises a truncated outer membrane protein pore, a flexible tether, a protein receptor and a peptide adaptor. When a protein ligand present in solution binds to the receptor, reversible capture and release events of the receptor can be measured as current transitions between two open substates of the pore. Notably, the binding and release of the receptor by a protein ligand can be unambiguously discriminated in a complex sample containing fetal bovine serum. Our selective nanopore sensor could be applied for single-molecule protein detection, could form the basis for a nanoproteomics platform or might be adapted to build tools for protein profiling and biomarker discovery.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Measuring high-affinity PPIs using a nanopore sensor.
Figure 2: Measuring low-affinity PPIs using a nanopore sensor.
Figure 3: Concurrent detection of weak and strong PPIs using a nanopore sensor.
Figure 4: Single-molecule protein detection and observation of transient PPIs using a nanopore sensor in FBS.

Accession codes


Protein Data Bank


  1. 1

    Hayes, S., Malacrida, B., Kiely, M. & Kiely, P.A. Studying protein-protein interactions: progress, pitfalls and solutions. Biochem. Soc. Trans. 44, 994–1004 (2016).

    CAS  Article  Google Scholar 

  2. 2

    Yoo, J., Lee, T.S., Choi, B., Shon, M.J. & Yoon, T.Y. Observing extremely weak protein-protein interactions with conventional single-molecule fluorescence microscopy. J. Am. Chem. Soc. 138, 14238–14241 (2016).

    CAS  Article  Google Scholar 

  3. 3

    De Keersmaecker, H. et al. Mapping transient protein interactions at the nanoscale in living mammalian cells. ACS Nano 12, 9842–9854 (2018).

    CAS  Article  Google Scholar 

  4. 4

    Nogal, B., Bowman, C.A. & Ward, A.B. Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level. J. Biol. Chem. 292, 19400–19410 (2017).

    CAS  Article  Google Scholar 

  5. 5

    Gonzalez, L.C. Protein microarrays, biosensors, and cell-based methods for secretome-wide extracellular protein-protein interaction mapping. Methods 57, 448–458 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Douzi, B. Protein-protein interactions: surface plasmon resonance. Methods Mol. Biol. 1615, 257–275 (2017).

    Article  Google Scholar 

  7. 7

    Pierce, M.M., Raman, C.S. & Nall, B.T. Isothermal titration calorimetry of protein-protein interactions. Methods 19, 213–221 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Sackmann, B. & Neher, E. Single-Channel Recording. Second edn. (Kluwer Academic/Plenum, New York, 1995).

  9. 9

    Wei, R., Gatterdam, V., Wieneke, R., Tampé, R. & Rant, U. Stochastic sensing of proteins with receptor-modified solid-state nanopores. Nat. Nanotechnol. 7, 257–263 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Ying, Y.L., Yu, R.J., Hu, Y.X., Gao, R. & Long, Y.T. Single antibody-antigen interactions monitored via transient ionic current recording using nanopore sensors. Chem. Commun. (Camb.) 53, 8620–8623 (2017).

    CAS  Article  Google Scholar 

  11. 11

    Weichbrodt, C. et al. Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms. Analyst 140, 4874–4881 (2015).

    CAS  Article  Google Scholar 

  12. 12

    Reiner, J.E. et al. Disease detection and management via single nanopore-based sensors. Chem. Rev. 112, 6431–6451 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat. Biotechnol. 34, 518–524 (2016).

    CAS  Article  Google Scholar 

  14. 14

    Ayub, M. & Bayley, H. Engineered transmembrane pores. Curr. Opin. Chem. Biol. 34, 117–126 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Burns, J.R., Seifert, A., Fertig, N. & Howorka, S. A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane. Nat. Nanotechnol. 11, 152–156 (2016).

    CAS  Article  Google Scholar 

  16. 16

    Howorka, S. Building membrane nanopores. Nat. Nanotechnol. 12, 619–630 (2017).

    CAS  Article  Google Scholar 

  17. 17

    Movileanu, L., Howorka, S., Braha, O. & Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat. Biotechnol. 18, 1091–1095 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Rotem, D., Jayasinghe, L., Salichou, M. & Bayley, H. Protein detection by nanopores equipped with aptamers. J. Am. Chem. Soc. 134, 2781–2787 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Harrington, L., Cheley, S., Alexander, L.T., Knapp, S. & Bayley, H. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc. Natl. Acad. Sci. USA 110, E4417–E4426 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Thakur, A.K., Larimi, M.G., Gooden, K. & Movileanu, L. Aberrantly large single-channel conductance of polyhistidine arm-containing protein nanopores. Biochemistry 56, 4895–4905 (2017).

    CAS  Article  Google Scholar 

  21. 21

    Locher, K.P. et al. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 95, 771–778 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Schreiber, G. & Fersht, A.R. Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. Biochemistry 32, 5145–5150 (1993).

    CAS  Article  Google Scholar 

  23. 23

    Schreiber, G. & Fersht, A.R. Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J. Mol. Biol. 248, 478–486 (1995).

    CAS  PubMed  Google Scholar 

  24. 24

    Deyev, S.M., Waibel, R., Lebedenko, E.N., Schubiger, A.P. & Plückthun, A. Design of multivalent complexes using the barnase*barstar module. Nat. Biotechnol. 21, 1486–1492 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Kudlinzki, D., Schmitt, A., Christian, H. & Ficner, R. Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22. Biol. Chem. 393, 1131–1140 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Mohammad, M.M., Howard, K.R. & Movileanu, L. Redesign of a plugged beta-barrel membrane protein. J. Biol. Chem. 286, 8000–8013 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Mohammad, M.M. et al. Engineering a rigid protein tunnel for biomolecular detection. J. Am. Chem. Soc. 134, 9521–9531 (2012).

    CAS  Article  Google Scholar 

  28. 28

    Perkins, J.R., Diboun, I., Dessailly, B.H., Lees, J.G. & Orengo, C. Transient protein-protein interactions: structural, functional, and network properties. Structure 18, 1233–1243 (2010).

    CAS  Article  Google Scholar 

  29. 29

    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 

  30. 30

    Kennedy, E., Dong, Z., Tennant, C. & Timp, G. Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore. Nat. Nanotechnol. 11, 968–976 (2016).

    CAS  Article  Google Scholar 

  31. 31

    Sze, J.Y.Y., Ivanov, A.P., Cass, A.E.G. & Edel, J.B. Single molecule multiplexed nanopore protein screening in human serum using aptamer modified DNA carriers. Nat. Commun. 8, 1552 (2017).

    Article  Google Scholar 

  32. 32

    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).

    Article  Google Scholar 

  33. 33

    Restrepo-Pérez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechnol. 13, 786–796 (2018).

    Article  Google Scholar 

  34. 34

    Buckle, A.M., Schreiber, G. & Fersht, A.R. Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. Biochemistry 33, 8878–8889 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Guillet, V., Lapthorn, A., Hartley, R.W. & Mauguen, Y. Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar. Structure 1, 165–176 (1993).

    CAS  Article  Google Scholar 

  36. 36

    McManus, O.B. & Magleby, K.L. Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle. J. Physiol. (Lond.) 402, 79–120 (1988).

    CAS  Article  Google Scholar 

Download references


We thank S. Loh for generosity in using his FPLC instrument in the very early stages of these studies and A. Matouschek (University of Texas at Austin) for his kindness in offering plasmids containing genes that encode Bn and Bs proteins, as well as M.L. Ghahari and M.M. Mohammad for their assistance in the very early stages of this project. This work was supported by US National Institutes of Health grants GM088403 (L.M.) and GM129429 (L.M.).

Author information




A.K.T. and L.M. designed research. A.K.T. performed research and analyzed data. A.K.T. and L.M. wrote the paper.

Corresponding author

Correspondence to Liviu Movileanu.

Ethics declarations

Competing interests

A.K.T. and L.M. are named inventors on two provisional patent applications (US 62/720,190 and US 62/579,982) filed by Syracuse University on this work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–8 (PDF 6811 kb)

Life Sciences Reporting Summary (PDF 131 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Thakur, A., Movileanu, L. Real-time measurement of protein–protein interactions at single-molecule resolution using a biological nanopore. Nat Biotechnol 37, 96–101 (2019).

Download citation

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