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Protein Sensing with Engineered Protein Nanopores

  • Mohammad M. Mohammad
  • Liviu MovileanuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 870)

Abstract

The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin (αHL) protein pore has been used extensively for the detection, characterization, and analysis of polypeptides because this protein nanopore is highly robust, versatile, and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins.

Key words

α-Hemolysin Nanopore Single-channel electrical recordings Protein engineering Biotechnology and biosensors 

Notes

Acknowledgments

This work was supported in part by grants from the US National Science Foundation (DMR-1006332) and National Institutes of Health (R01 GM088403) to LM.

References

  1. 1.
    Kasianowicz JJ, Brandin E, Branton D, Deamer DW (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A 93: 13770–13773CrossRefGoogle Scholar
  2. 2.
    Bayley H (2009) Membrane-protein structure piercing insights. Nature 459:651–652CrossRefGoogle Scholar
  3. 3.
    Bayley H, Cremer PS (2001) Stochastic sensors inspired by biology. Nature 413:226–230CrossRefGoogle Scholar
  4. 4.
    Movileanu L (2009) Interrogating single proteins through nanopores: challenges and opportunities. Trends Biotechnol 27:333–341CrossRefGoogle Scholar
  5. 5.
    Movileanu L (2008) Squeezing a single polypeptide through a nanopore. Soft Matter 4:925–931CrossRefGoogle Scholar
  6. 6.
    Howorka S, Siwy Z (2009) Nanopore analytics: sensing of single molecules. Chem Soc Rev 38:2360–2384CrossRefGoogle Scholar
  7. 7.
    Siwy ZS, Howorka S (2010) Engineered voltage-responsive nanopores. Chem Soc Rev 39:1115–1132CrossRefGoogle Scholar
  8. 8.
    Branton D, Deamer DW, Marziali A, Bayley H, Benner SA, Butler T, Di Ventra M, Garaj S, Hibbs A, Huang XH, Jovanovich SB, Krstic PS, Lindsay S, Ling XSS, Mastrangelo CH, Meller A, Oliver JS, Pershin YV, Ramsey JM, Riehn R, Soni GV, Tabard-Cossa V, Wanunu M, Wiggin M, Schloss JA (2008) The potential and challenges of nanopore sequencing. Nat Biotechnol 26:1146–1153CrossRefGoogle Scholar
  9. 9.
    Song LZ, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274:1859–1866CrossRefGoogle Scholar
  10. 10.
    Movileanu L, Cheley S, Howorka S, Braha O, Bayley H (2001) Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules. J Gen Physiol 117:239–251CrossRefGoogle Scholar
  11. 11.
    Wolfe AJ, Mohammad MM, Cheley S, Bayley H, Movileanu L (2007) Catalyzing the translocation of polypeptides through attractive interactions. J Am Chem Soc 129:14034–14041CrossRefGoogle Scholar
  12. 12.
    Movileanu L, Howorka S, Braha O, Bayley H (2000) Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat Biotechnol 18:1091–1095CrossRefGoogle Scholar
  13. 13.
    Movileanu L, Bayley H (2001) Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law. Proc Natl Acad Sci U S A 98:10137–10141CrossRefGoogle Scholar
  14. 14.
    Howorka S, Movileanu L, Lu XF, Magnon M, Cheley S, Braha O, Bayley H (2000) A protein pore with a single polymer chain tethered within the lumen. J Am Chem Soc 122: 2411–2416CrossRefGoogle Scholar
  15. 15.
    Bezrukov SM, Vodyanoy I, Brutyan RA, Kasianowicz JJ (1996) Dynamics and free energy of polymers partitioning into a nanoscale pore. Macromolecules 29:8517–8522CrossRefGoogle Scholar
  16. 16.
    Bezrukov SM, Kasianowicz JJ (1993) Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel. Phys Rev Lett 70:2352–2355CrossRefGoogle Scholar
  17. 17.
    Krasilnikov OV, Rodrigues CG, Bezrukov SM (2006) Single polymer molecules in a protein nanopore in the limit of a strong polymer-pore attraction. Phys Rev Lett 97: issue 1 article 018301Google Scholar
  18. 18.
    Jung Y, Bayley H, Movileanu L (2006) Temperature-responsive protein pores. J Am Chem Soc 128:15332–15340CrossRefGoogle Scholar
  19. 19.
    Kang XF, Gu LQ, Cheley S, Bayley H (2005) Single protein pores containing molecular adapters at high temperatures. Angew Chem Int Ed 44:1495–1499CrossRefGoogle Scholar
  20. 20.
    Goodrich CP, Kirmizialtin S, Huyghues-Despointes BM, Zhu AP, Scholtz JM, Makarov DE, Movileanu L (2007) Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations. J Phys Chem B 111:3332–3335CrossRefGoogle Scholar
  21. 21.
    Movileanu L, Schmittschmitt JP, Scholtz JM, Bayley H (2005) Interactions of peptides with a protein pore. Biophys J 89: 1030–1045CrossRefGoogle Scholar
  22. 22.
    Oukhaled G, Mathe J, Biance AL, Bacri L, Betton JM, Lairez D, Pelta J, Auvray L (2007) Unfolding of proteins and long transient conformations detected by single nanopore recording. Phys Rev Lett 98: issue 15 article 158101Google Scholar
  23. 23.
    Pastoriza-Gallego M, Oukhaled G, Mathe J, Thiebot B, Betton JM, Auvray L, Pelta J (2007) Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry. FEBS Lett 581:3371–3376CrossRefGoogle Scholar
  24. 24.
    Bezrukov SM (2000) Ion channels as molecular Coulter counters to probe metabolite transport. J Membr Biol 174:1–13CrossRefGoogle Scholar
  25. 25.
    Geissler A, Chacinska A, Truscott KN, Wiedemann N, Brandner K, Sickmann A, Meyer HE, Meisinger C, Pfanner N, Rehling P (2002) The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 111:507–518CrossRefGoogle Scholar
  26. 26.
    Mohammad MM, Movileanu L (2008) Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology. Eur Biophys J Biophys Lett 37:913–925CrossRefGoogle Scholar
  27. 27.
    Mohammad MM, Prakash S, Matouschek A, Movileanu L (2008) Controlling a single protein in a nanopore through electrostatic traps. J Am Chem Soc 130:4081–4088CrossRefGoogle Scholar
  28. 28.
    Walker B, Krishnasastry M, Zorn L, Bayley H (1992) Assembly of the oligomeric membrane pore formed by staphylococcal alpha-hemolysin examined by truncation mutagenesis. J Biol Chem 267:21782–21786Google Scholar
  29. 29.
    Gouaux JE, Braha O, Hobaugh MR, Song LZ, Cheley S, Shustak C, Bayley H (1994) Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore. Proc Natl Acad Sci U S A 91:12828–12831CrossRefGoogle Scholar
  30. 30.
    Stefureac R, Long YT, Kraatz HB, Howard P, Lee JS (2006) Transport of alpha-helical peptides through alpha-hemolysin and aerolysin pores. Biochemistry 45:9172–9179CrossRefGoogle Scholar
  31. 31.
    Maglia G, Restrepo MR, Mikhailova E, Bayley H (2008) Enhanced translocation of single DNA molecules through alpha-hemolysin nanopores by manipulation of internal charge. Proc Natl Acad Sci U S A 105: 19720–19725CrossRefGoogle Scholar
  32. 32.
    Miles G, Movileanu L, Bayley H (2002) Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore. Protein Sci 11:894–902CrossRefGoogle Scholar
  33. 33.
    Movileanu L, Cheley S, Bayley H (2003) Partitioning of individual flexible polymers into a nanoscopic protein pore. Biophys J 85: 897–910CrossRefGoogle Scholar
  34. 34.
    Zhao Q, Sigalov G, Dimitrov V, Dorvel B, Mirsaidov U, Sligar S, Aksimentiev A, Timp G (2007) Detecting SNPs using a synthetic nanopore. Nano Lett 7:1680–1685CrossRefGoogle Scholar
  35. 35.
    Butler TZ, Pavlenok M, Derrington IM, Niederweis M, Gundlach JH (2008) Single-molecule DNA detection with an engineered MspA protein nanopore. Proc Natl Acad Sci U S A 105:20647–20652CrossRefGoogle Scholar
  36. 36.
    Guiard B (1985) Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome-C oxidoreductase (cytochrome-B2). EMBO J 4:3265–3272Google Scholar
  37. 37.
    Matouschek A, Azem A, Ratliff K, Glick BS, Schmid K, Schatz G (1997) Active unfolding of precursor proteins during mitochondrial protein import. EMBO J 16: 6727–6736CrossRefGoogle Scholar
  38. 38.
    Shim JW, Yang M, Gu LQ (2007) In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv. FEBS Lett 581:1027–1034CrossRefGoogle Scholar
  39. 39.
    Kadurugamuwa JL, Beveridge TJ (1995) Virulence factors are released from pseudomonas-aeruginosa in association with membrane-vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme-secretion. J Bacteriol 177:3998–4008Google Scholar
  40. 40.
    Mohammad M, York RD, Hommel J, Kapler GM (2003) Characterization of a novel origin recognition complex-like complex: implications for DNA recognition, cell cycle control, and locus-specific gene amplification. Mol Cell Biol 23:5005–5017CrossRefGoogle Scholar
  41. 41.
    Mohammad M, Saha S, Kapler GM (2000) Three different proteins recognize a multifunctional determinant that controls replication initiation, fork arrest and transcription in Tetrahymena. Nucleic Acids Res 28: 843–851CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of PhysicsSyracuse UniversitySyracuseUSA

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