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Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore

Nature Biotechnology volume 18, pages 1091–1095 (2000)Cite this article

Abstract

Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.

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Figure 1: Preparation and analysis of heteroheptameric αHL pores.
Figure 2: Response of H6106C-PEG-biotin1 to WT and W120A streptavidins and a mouse anti-biotin mAb.
Figure 3: Kinetic model of the interactions between the αHL pore H6106C-PEG-biotin1 and streptavidin at the cis and trans sides of the bilayer.

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References

  1. Heath, J.R., Kuekes, P.J., Snider, G.S. & Williams, R.S. A defect-tolerant computer architecture: opportunities for nanotechnology . Science 280, 1716–1721 (1998).

    Article  CAS  Google Scholar 

  2. Davis, A.P. Synthetic molecular motors. Nature 401, 120–121 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Seeman, N.C. DNA engineering and its applications to nanotechnology. Trends Biotechnol. 17, 437–443 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Song, L. et al. Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science 274, 1859– 1865 (1996).

    Article  CAS  PubMed  Google Scholar 

  5. Gouaux, E. α-Hemolysin from Staphylococcus aureus: an archetype of β-barrel, channel-forming toxins. J. Struct. Biol. 121, 110–122 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Chang, C.-Y., Niblack, B., Walker, B. & Bayley, H. A photogenerated pore-forming protein. Chem. Biol. 2, 391 –400 (1995).

    Article  CAS  PubMed  Google Scholar 

  7. Panchal, R.G., Cusack, E., Cheley, S. & Bayley, H. Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat. Biotechnol. 14, 852–856 ( 1996).

    Article  CAS  PubMed  Google Scholar 

  8. Russo, M.J., Bayley, H. & Toner, M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat. Biotechnol. 15, 278–282 (1997).

    Article  CAS  PubMed  Google Scholar 

  9. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497–505 ( 1997).

    Article  CAS  PubMed  Google Scholar 

  10. Gu, L.-Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Bezrukov, S.M., Vodyanoy, I. & Parsegian, V.A. Counting polymers moving through a single ion channel . Nature 370, 279–281 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. Bezrukov, S.M., Vodyanoy, I., Brutyan, R.A. & Kasianowicz, J.J. Dynamics and free energy of polymer partitioning into a nanoscale pore. Macromolecules 29, 8517–8522 (1996).

    Article  CAS  Google Scholar 

  13. Merzlyak, P.G. et al. Polymeric nonelectrolytes to probe pore geometry: application to the α-toxin transmembrane channel. Biophys. J. 77, 3023–3033 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kasianowicz, J.J., Brandin, E., Branton, D. & Deamer, D.W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. USA 93, 13770– 13773 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Akeson, M., Branton, D., Kasianowicz, J.J., Brandin, E. & Deamer, D.W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227–3233 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Howorka, S. et al. A protein pore with a single polymer chain tethered within the lumen. J. Am. Chem. Soc. 122, 2411– 2416 (2000).

    Article  CAS  Google Scholar 

  17. Sano, T. & Cantor, C.R. Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc. Natl. Acad. Sci. USA 92, 3180–3184 ( 1995).

    Article  CAS  PubMed  Google Scholar 

  18. Chilkoti, A., Tan, P.H. & Stayton, P.S. Site-directed mutagenesis studies of the high-affinity streptavidin–biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc. Natl. Acad. Sci. USA 92, 1754–1758 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Chilkoti, A., Boland, T., Ratner, B.D. & Stayton, P.S. The relationship between ligand-binding thermodynamics and protein–ligand interaction forces measured by atomic force microscopy. Biophys. J. 69, 2125–2130 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pérez-Luna, V.H. et al. Molecular recognition between genetically engineered streptavidin and surface-bound biotin. J. Am. Chem. Soc. 121, 6469–6478 (1999).

    Article  Google Scholar 

  21. Slatin, S.L., Qiu, X.-Q., Jakes, K.S. & Finkelstein, A. Identification of a translocated protein segment in a voltage-dependent channel. Nature 371, 158–161 ( 1994).

    Article  CAS  PubMed  Google Scholar 

  22. Wong, J.Y., Kuhl, T.L., Israelachvili, J.N., Mullah, N. & Zalipsky, S. Direct measurement of a tethered ligand–receptor interaction potential. Science 275, 820–822 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Bayley, H., Braha, O. & Gu, L.-Q. Stochastic sensing with protein pores. Adv. Mater. 12, 139–142 ( 2000).

    Article  CAS  Google Scholar 

  24. Myszka, D.G. Improving biosensor design. J. Mol. Recognition 12, 279–284 (1999).

    Article  CAS  Google Scholar 

  25. Mao, C., Sun, W., Shen, Z. & Seeman, N.C. A nanomechanical device based on the B-Z transition of DNA. Nature 397 , 144–146 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Mehta, A.D., Rief, M., Spudich, J.A., Smith, D.A. & Simmons, R.M. Single-molecule biomechanics with optical methods . Science 283, 1689–1695 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Xie, X.S. & Lu, H.P. Single-molecule enzymology. J. Biol. Chem. 274, 15967–15970 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Marszalek, P.E. et al. Mechanical unfolding intermediates in titin molecules. Nature 402, 100–103 ( 1999).

    Article  CAS  PubMed  Google Scholar 

  30. Stayton, P.S. et al. Control of protein–ligand recognition using a stimuli-responsive polymer. Nature 378, 472– 474 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Walker, B.J. & Bayley, H. A pore-forming protein with a protease-activated trigger. Protein Eng. 7, 91– 97 (1994).

    Article  CAS  PubMed  Google Scholar 

  32. Walker, B. & Bayley, H. Key residues for membrane binding, oligomerization, and pore-forming activity of staphylococcal α-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification . J. Biol. Chem. 270, 23065– 23071 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Cheley, S., Braha, O., Lu, X., Conlan, S., & Bayley, H. A functional protein pore with a “retro” transmembrane domain. Protein Sci. 8, 1257 –1267 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Walker, B.J., Krishnasastry, M., Zorn, L., Kasianowicz, J.J. & Bayley, H. Functional expression of the α-hemolysin of Staphylococcus aureus in intact Escherichia coli and in cell lysates. J. Biol. Chem. 267, 10902– 10909 (1992).

    CAS  PubMed  Google Scholar 

  35. Walker, B. & Bayley, H. Restoration of pore-forming activity in staphylococcal α-hemolysin by targeted chemical modification. Protein Eng. 8, 491–495 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Montal, M. & Mueller, P. Formation of bimolecular membranes from lipid monolayers and study of their electrical properties. Proc. Natl. Acad. Sci. USA 69, 3561– 3566 (1972).

    Article  CAS  PubMed  Google Scholar 

  37. Christopher, J.A. SPOCK: the structural properties observation and calculation kit (program manual). (Center for Macromolecular Design, Texas A&M University, College Station, TX; 1998).

    Google Scholar 

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Acknowledgements

We are most grateful to Pat Stayton and David Hyre for the W120A protein. This work was supported by a Multidisciplinary University Research Initiative (MURI) award (Office of Naval Research) to H.B., a MURI award (Air Force Office of Scientific Research) to A.J. Welch, the Department of Energy and the Texas Advanced Technology Program. S.H. holds a postdoctoral fellowship from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung). We thank Li-Qun Gu, Yong Zhang, and Sean Conlan for their help and suggestions.

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Author notes

  1. Liviu Movileanu and Stefan Howorka: These two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX, 77843-1114

    Liviu Movileanu, Stefan Howorka, Orit Braha & Hagan Bayley

  2. Department of Chemistry, Texas A&M University, College Station, TX, 77843-3255

    Hagan Bayley

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  1. Liviu Movileanu
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Correspondence to Hagan Bayley.

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Movileanu, L., Howorka, S., Braha, O. et al. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat Biotechnol 18, 1091–1095 (2000). https://doi.org/10.1038/80295

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