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

Abstract

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.

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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.

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Acknowledgements

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.).

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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.

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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.

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

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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). https://doi.org/10.1038/nbt.4316

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