A monodisperse transmembrane α-helical peptide barrel


The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

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Figure 1: Structure of E. coli Wza, peptide design and electrical properties of the cWza pore.
Figure 2: Electrical properties of mutant cWza pores.
Figure 3: Interaction of cWza peptide pores with cationic CDs.
Figure 4: Orientation and stoichiometry of the cWza-Y373C pore.
Figure 5: CD-templated barrel and its electrical properties.
Figure 6: Model for membrane insertion and pore formation by cWza peptides.

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Protein Data Bank


  1. 1

    Woolfson, D. N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005).

  2. 2

    Woolfson, D. N. et al. De novo protein design: how do we expand into the universe of possible protein structures? Curr. Opin. Struct. Biol. 33, 16–26 (2015).

  3. 3

    Woolfson, D. N., Bartlett, G. J., Bruning, M. & Thomson, A. R. New currency for old rope: from coiled-coil assemblies to alpha-helical barrels. Curr. Opin. Struct. Biol. 22, 432–441 (2012).

  4. 4

    Lear, J. D., Wasserman, Z. R. & DeGrado, W. F. Synthetic amphiphilic peptide models for protein ion channels. Science 240, 1177–1181 (1988).

  5. 5

    Joh, N. H. et al. De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 346, 1520–1524 (2014).

  6. 6

    Franceschini, L., Soskine, M., Biesemans, A. & Maglia, G. A nanopore machine promotes the vectorial transport of DNA across membranes. Nat. Commun. 4, 2415 (2013).

  7. 7

    Bayley, H. Membrane-protein structure: piercing insights. Nature 459, 651–652 (2009).

  8. 8

    Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).

  9. 9

    Kong, L. et al. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor. Nat. Chem. 5, 651–659 (2013).

  10. 10

    Soskine, M. et al. An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry. Nano Lett. 12, 4895–4900 (2012).

  11. 11

    Soskine, M., Biesemans, A., De Maeyer, M. & Maglia, G. Tuning the size and properties of ClyA nanopores assisted by directed evolution. J. Am. Chem. Soc. 135, 13456–13463 (2013).

  12. 12

    Tanaka, K., Caaveiro, J. M., Morante, K., González-Mañas, J. M. & Tsumoto, K. Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat. Commun. 6, 6337 (2015).

  13. 13

    Zaccai, N. R. et al. A de novo peptide hexamer with a mutable channel. Nat. Chem. Biol. 7, 935–941 (2011).

  14. 14

    Thomson, A. R. et al. Computational design of water-soluble alpha-helical barrels. Science 346, 485–488 (2014).

  15. 15

    Bayley, H. Designed membrane channels and pores. Curr. Opin. Biotechnol. 10, 94–103 (1999).

  16. 16

    Bayley, H. & Jayasinghe, L. Functional engineered channels and pores (Review). Mol. Membr. Biol. 21, 209–220 (2004).

  17. 17

    Majd, S. et al. Applications of biological pores in nanomedicine, sensing, and nanoelectronics. Curr. Opin. Biotechnol. 21, 439–476 (2010).

  18. 18

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

  19. 19

    Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226–230 (2001).

  20. 20

    Bayley, H. Nanopore sequencing: from imagination to reality. Clin. Chem. 61, 25–31 (2015).

  21. 21

    Jain, M. et al. Improved data analysis for the MinION nanopore sequencer. Nat. Methods 12, 351–356 (2015).

  22. 22

    Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).

  23. 23

    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).

  24. 24

    Banerjee, A. et al. Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores. Proc. Natl Acad. Sci. USA 107, 8165–8170 (2010).

  25. 25

    Walshaw, J. & Woolfson, D. N. Socket: a program for identifying and analysing coiled-coil motifs within protein structures. J. Mol. Biol. 307, 1427–1450 (2001).

  26. 26

    van den Berg, B., Prathyusha Bhamidimarri, S., Dahyabhai Prajapati, J., Kleinekathöfer, U. & Winterhalter, M. Outer-membrane translocation of bulky small molecules by passive diffusion. Proc. Natl Acad. Sci. USA 112, E2991–E2999 (2015).

  27. 27

    Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

  28. 28

    Mueller, M., Grauschopf, U., Maier, T., Glockshuber, R. & Ban, N. The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism. Nature 459, 726–730 (2009).

  29. 29

    Miles, G., Movileanu, L. & Bayley, H. Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore. Protein Sci. 11, 894–902 (2002).

  30. 30

    Smart, O. S., Breed, J., Smith, G. R. & Sansom, M. S. A novel method for structure-based prediction of ion channel conductance properties. Biophys. J. 72, 1109–1126 (1997).

  31. 31

    Sukharev, S., Betanzos, M., Chiang, C. S. & Guy, H. R. The gating mechanism of the large mechanosensitive channel MscL. Nature 409, 720–724 (2001).

  32. 32

    Wang, Y. et al. Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. eLife 3, e01834 (2014).

  33. 33

    Pliotas, C . et al. The role of lipids in mechanosensation. Nat. Struct. Mol. Biol. 22, 991–8 (2015).

  34. 34

    Walker, B., Krishnasastry, M., Zorn, L. & Bayley, H. Assembly of the oligomeric membrane pore formed by staphylococcal alpha-hemolysin examined by truncation mutagenesis. J. Biol. Chem. 267, 21782–6 (1992).

  35. 35

    Walker, B., Braha, O., Cheley, S. & Bayley, H. An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch. Chem. Biol. 2, 99–105 (1995).

  36. 36

    Dunstone, M. A. & Tweten, R. K. Packing a punch: the mechanism of pore formation by cholesterol dependent cytolysins and membrane attack complex/perforin-like proteins. Curr. Opin. Struct. Biol. 22, 342–349 (2012).

  37. 37

    Leung, C. et al. Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin. eLife 3, e04247 (2014).

  38. 38

    Stoddart, D. et al. Functional truncated membrane pores. Proc. Natl Acad. Sci. USA 111, 2425–2430 (2014).

  39. 39

    Karginov, V. A. Cyclodextrin derivatives as anti-infectives. Curr. Opin. Pharmacol. 13, 717–725 (2013).

  40. 40

    Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250 (2005).

  41. 41

    Cirac, A. D. et al. The molecular basis for antimicrobial activity of pore-forming cyclic peptides. Biophys. J. 100, 2422–2431 (2011).

  42. 42

    Song, C. et al. Crystal structure and functional mechanism of a human antimicrobial membrane channel. Proc. Natl Acad. Sci. USA 110, 4586–4591 (2013).

  43. 43

    Haswell, E. S., Phillips, R. & Rees, D. C. Mechanosensitive channels: what can they do and how do they do it? Structure 19, 1356–1369 (2011).

  44. 44

    Naismith, J. H. & Booth, I. R. Bacterial mechanosensitive channels—MscS: evolution's solution to creating sensitivity in function. Annu. Rev. Biophys. 41, 157–177 (2012).

  45. 45

    Lee, J. & Bayley, H. Semisynthetic protein nanoreactor for single-molecule chemistry. Proc. Natl Acad. Sci. USA 112, 13768–13773 (2015).

  46. 46

    Fernandez-Lopez, S. et al. Antibacterial agents based on the cyclic D,L-alpha-peptide architecture. Nature 412, 452–455 (2001).

  47. 47

    Fjell, C. D., Hiss, J. A., Hancock, R. E. & Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug. Discov. 11, 37–51 (2012).

  48. 48

    Hoskin, D. W. & Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta 1778, 357–375 (2008).

  49. 49

    Gaspar, D., Veiga, A. S. & Castanho, M. A. From antimicrobial to anticancer peptides. A review. Front. Microbiol. 4, 294 (2013).

  50. 50

    Mantri, S., Tanuj Sapra, K., Cheley, S., Sharp, T. H. & Bayley, H. An engineered dimeric protein pore that spans adjacent lipid bilayers. Nat. Commun. 4, 1725 (2013).

  51. 51

    Gutsmann, T., Heimburg, T., Keyser, U., Mahendran, K. R. & Winterhalter, M. Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat. Protoc. 10, 188–198 (2015).

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The authors acknowledge a Biotechnology and Biological Sciences Research Council grant (BB/J009784/1) and the European Research Council (340764) for financial support. D.N.W. holds a Royal Society Wolfson Research Merit Award.

Author information

K.R.M. performed and analysed the current recordings. A.N. synthesized peptides and determined their biophysical properties. L.K. produced the Wza protein and synthesized the CD derivatives. A.N. and A.R.T. performed the molecular modelling. A.N. and R.B.S. performed the molecular dynamics simulations. K.R.M., A.N., D.N.W. and H.B. designed experiments and wrote the paper.

Correspondence to Derek N. Woolfson or Hagan Bayley.

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Mahendran, K., Niitsu, A., Kong, L. et al. A monodisperse transmembrane α-helical peptide barrel. Nature Chem 9, 411–419 (2017) doi:10.1038/nchem.2647

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