An Engineered ClyA Nanopore Detects Folded Target Proteins by Selective External Association and Pore Entry

Overview

Journal Nano Lett

Publisher American Chemical Society

Specialty Biotechnology

Date 2012 Aug 2

PMID 22849517

Citations 87

Authors

Misha Soskine

Annemie Biesemans

Benjamien Moeyaert

Stephen Cheley

Hagan Bayley

Giovanni Maglia

Affiliations

Soon will be listed here.

Abstract

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.

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References

Fologea D, Ledden B, McNabb D, Li J . Electrical characterization of protein molecules by a solid-state nanopore. Appl Phys Lett. 2008; 91(5):539011-539013. PMC: 2288568. DOI: 10.1063/1.2767206. View

Bilokapic S, Schwartz T . 3D ultrastructure of the nuclear pore complex. Curr Opin Cell Biol. 2012; 24(1):86-91. PMC: 3398480. DOI: 10.1016/j.ceb.2011.12.011. View

Tainsky M . Genomic and proteomic biomarkers for cancer: a multitude of opportunities. Biochim Biophys Acta. 2009; 1796(2):176-93. PMC: 2752479. DOI: 10.1016/j.bbcan.2009.04.004. View

Grossman E, Medalia O, Zwerger M . Functional architecture of the nuclear pore complex. Annu Rev Biophys. 2012; 41:557-84. DOI: 10.1146/annurev-biophys-050511-102328. View

Howorka S, Nam J, Bayley H, Kahne D . Stochastic detection of monovalent and bivalent protein-ligand interactions. Angew Chem Int Ed Engl. 2004; 43(7):842-6. DOI: 10.1002/anie.200352614. View

Desmet J, De Maeyer M, Hazes B, Lasters I . The dead-end elimination theorem and its use in protein side-chain positioning. Nature. 2011; 356(6369):539-42. DOI: 10.1038/356539a0. View

Wei R, Gatterdam V, Wieneke R, Tampe R, Rant U . Stochastic sensing of proteins with receptor-modified solid-state nanopores. Nat Nanotechnol. 2012; 7(4):257-63. DOI: 10.1038/nnano.2012.24. View

Hammerstein A, Shin S, Bayley H . Single-molecule kinetics of two-step divalent cation chelation. Angew Chem Int Ed Engl. 2010; 49(30):5085-90. DOI: 10.1002/anie.200906601. View

Riaz S, Alam S, Akhtar M . Proteomic identification of human serum biomarkers in diabetes mellitus type 2. J Pharm Biomed Anal. 2009; 51(5):1103-7. DOI: 10.1016/j.jpba.2009.11.016. View

10.

Firnkes M, Pedone D, Knezevic J, Doblinger M, Rant U . Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis. Nano Lett. 2010; 10(6):2162-7. DOI: 10.1021/nl100861c. View

11.

Astier Y, Kainov D, Bayley H, Tuma R, Howorka S . Stochastic detection of motor protein-RNA complexes by single-channel current recording. Chemphyschem. 2007; 8(15):2189-94. DOI: 10.1002/cphc.200700179. View

12.

Gu L, Cheley S, Bayley H . Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore. Proc Natl Acad Sci U S A. 2003; 100(26):15498-503. PMC: 307596. DOI: 10.1073/pnas.2531778100. View

13.

Cheley S, Xie H, Bayley H . A genetically encoded pore for the stochastic detection of a protein kinase. Chembiochem. 2006; 7(12):1923-7. DOI: 10.1002/cbic.200600274. View

14.

Bayley H, Cremer P . Stochastic sensors inspired by biology. Nature. 2001; 413(6852):226-30. DOI: 10.1038/35093038. View

15.

Beck M, Lucic V, Forster F, Baumeister W, Medalia O . Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature. 2007; 449(7162):611-5. DOI: 10.1038/nature06170. View

16.

Yusko E, Johnson J, Majd S, Prangkio P, Rollings R, Li J . Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat Nanotechnol. 2011; 6(4):253-60. PMC: 3071889. DOI: 10.1038/nnano.2011.12. View

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. 2000; 18(10):1091-5. DOI: 10.1038/80295. View

18.

Eifler N, Vetsch M, Gregorini M, Ringler P, Chami M, Philippsen A . Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state. EMBO J. 2006; 25(11):2652-61. PMC: 1478193. DOI: 10.1038/sj.emboj.7601130. View

19.

Wu H, Bayley H . Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore. J Am Chem Soc. 2008; 130(21):6813-9. DOI: 10.1021/ja8004607. View

20.

Braha O, Gu L, Zhou L, Lu X, Cheley S, Bayley H . Simultaneous stochastic sensing of divalent metal ions. Nat Biotechnol. 2000; 18(9):1005-7. DOI: 10.1038/79275. View