Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jun;8(6):1257–1267. doi: 10.1110/ps.8.6.1257

A functional protein pore with a "retro" transmembrane domain.

S Cheley 1, O Braha 1, X Lu 1, S Conlan 1, H Bayley 1
PMCID: PMC2144353  PMID: 10386875

Abstract

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bhakdi S., Füssle R., Tranum-Jensen J. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5475–5479. doi: 10.1073/pnas.78.9.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhakdi S., Muhly M., Füssle R. Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin. Infect Immun. 1984 Nov;46(2):318–323. doi: 10.1128/iai.46.2.318-323.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braha O., Walker B., Cheley S., Kasianowicz J. J., Song L., Gouaux J. E., Bayley H. Designed protein pores as components for biosensors. Chem Biol. 1997 Jul;4(7):497–505. doi: 10.1016/s1074-5521(97)90321-5. [DOI] [PubMed] [Google Scholar]
  4. Chang C. Y., Niblack B., Walker B., Bayley H. A photogenerated pore-forming protein. Chem Biol. 1995 Jun;2(6):391–400. doi: 10.1016/1074-5521(95)90220-1. [DOI] [PubMed] [Google Scholar]
  5. Cheley S., Malghani M. S., Song L., Hobaugh M., Gouaux J. E., Yang J., Bayley H. Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel. Protein Eng. 1997 Dec;10(12):1433–1443. doi: 10.1093/protein/10.12.1433. [DOI] [PubMed] [Google Scholar]
  6. Chorev M., Goodman M. Recent developments in retro peptides and proteins--an ongoing topochemical exploration. Trends Biotechnol. 1995 Oct;13(10):438–445. doi: 10.1016/S0167-7799(00)88999-4. [DOI] [PubMed] [Google Scholar]
  7. Derossi D., Calvet S., Trembleau A., Brunissen A., Chassaing G., Prochiantz A. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem. 1996 Jul 26;271(30):18188–18193. doi: 10.1074/jbc.271.30.18188. [DOI] [PubMed] [Google Scholar]
  8. Füssle R., Bhakdi S., Sziegoleit A., Tranum-Jensen J., Kranz T., Wellensiek H. J. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. doi: 10.1083/jcb.91.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gouaux E. alpha-Hemolysin from Staphylococcus aureus: an archetype of beta-barrel, channel-forming toxins. J Struct Biol. 1998;121(2):110–122. doi: 10.1006/jsbi.1998.3959. [DOI] [PubMed] [Google Scholar]
  10. Gouaux J. E., Braha O., Hobaugh M. R., Song L., Cheley S., Shustak C., Bayley H. Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12828–12831. doi: 10.1073/pnas.91.26.12828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guptasarma P. Reversal of peptide backbone direction may result in the mirroring of protein structure. FEBS Lett. 1992 Oct 5;310(3):205–210. doi: 10.1016/0014-5793(92)81333-h. [DOI] [PubMed] [Google Scholar]
  12. Heinz D. W., Baase W. A., Matthews B. W. Folding and function of a T4 lysozyme containing 10 consecutive alanines illustrate the redundancy of information in an amino acid sequence. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3751–3755. doi: 10.1073/pnas.89.9.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hutchinson E. G., Sessions R. B., Thornton J. M., Woolfson D. N. Determinants of strand register in antiparallel beta-sheets of proteins. Protein Sci. 1998 Nov;7(11):2287–2300. doi: 10.1002/pro.5560071106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ido Y., Vindigni A., Chang K., Stramm L., Chance R., Heath W. F., DiMarchi R. D., Di Cera E., Williamson J. R. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science. 1997 Jul 25;277(5325):563–566. doi: 10.1126/science.277.5325.563. [DOI] [PubMed] [Google Scholar]
  15. Jones D. H., Howard B. H. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. Biotechniques. 1991 Jan;10(1):62–66. [PubMed] [Google Scholar]
  16. Kasianowicz J. J., Brandin E., Branton D., Deamer D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13770–13773. doi: 10.1073/pnas.93.24.13770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Korchev Y. E., Bashford C. L., Alder G. M., Kasianowicz J. J., Pasternak C. A. Low conductance states of a single ion channel are not 'closed'. J Membr Biol. 1995 Oct;147(3):233–239. doi: 10.1007/BF00234521. [DOI] [PubMed] [Google Scholar]
  18. Krasilnikov O. V., Sabirov R. Z., Ternovsky V. I., Merzliak P. G., Muratkhodjaev J. N. A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes. FEMS Microbiol Immunol. 1992 Sep;5(1-3):93–100. doi: 10.1111/j.1574-6968.1992.tb05891.x. [DOI] [PubMed] [Google Scholar]
  19. Krasilnikov O. V., Sabirov R. Z., Ternovsky V. I., Merzliak P. G., Tashmukhamedov B. A. The structure of Staphylococcus aureus alpha-toxin-induced ionic channel. Gen Physiol Biophys. 1988 Oct;7(5):467–473. [PubMed] [Google Scholar]
  20. Lacroix E., Viguera A. R., Serrano L. Reading protein sequences backwards. Fold Des. 1998;3(2):79–85. doi: 10.1016/S1359-0278(98)00013-3. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. MacKenzie K. R., Engelman D. M. Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3583–3590. doi: 10.1073/pnas.95.7.3583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matthews B. W., Weaver L. H., Kester W. R. The conformation of thermolysin. J Biol Chem. 1974 Dec 25;249(24):8030–8044. [PubMed] [Google Scholar]
  24. Menestrina G. Ionic channels formed by Staphylococcus aureus alpha-toxin: voltage-dependent inhibition by divalent and trivalent cations. J Membr Biol. 1986;90(2):177–190. doi: 10.1007/BF01869935. [DOI] [PubMed] [Google Scholar]
  25. Merrifield R. B., Juvvadi P., Andreu D., Ubach J., Boman A., Boman H. G. Retro and retroenantio analogs of cecropin-melittin hybrids. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3449–3453. doi: 10.1073/pnas.92.8.3449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Middaugh C. R., Thomson J. A., Burke C. J., Mach H., Naylor A. M., Bogusky M. J., Ryan J. A., Pitzenberger S. M., Ji H., Cordingley J. S. Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni. Protein Sci. 1993 Jun;2(6):900–914. doi: 10.1002/pro.5560020604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Milton R. C., Milton S. C., Kent S. B. Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Science. 1992 Jun 5;256(5062):1445–1448. doi: 10.1126/science.1604320. [DOI] [PubMed] [Google Scholar]
  28. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nicholson H., Söderlind E., Tronrud D. E., Matthews B. W. Contributions of left-handed helical residues to the structure and stability of bacteriophage T4 lysozyme. J Mol Biol. 1989 Nov 5;210(1):181–193. doi: 10.1016/0022-2836(89)90299-4. [DOI] [PubMed] [Google Scholar]
  30. Olson R., Nariya H., Yokota K., Kamio Y., Gouaux E. Crystal structure of staphylococcal LukF delineates conformational changes accompanying formation of a transmembrane channel. Nat Struct Biol. 1999 Feb;6(2):134–140. doi: 10.1038/5821. [DOI] [PubMed] [Google Scholar]
  31. Olszewski K. A., Kolinski A., Skolnick J. Does a backwardly read protein sequence have a unique native state? Protein Eng. 1996 Jan;9(1):5–14. doi: 10.1093/protein/9.1.5. [DOI] [PubMed] [Google Scholar]
  32. Panchal R. G., Cusack E., Cheley S., Bayley H. Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat Biotechnol. 1996 Jul;14(7):852–856. doi: 10.1038/nbt0796-852. [DOI] [PubMed] [Google Scholar]
  33. Regan L. What determines where alpha-helices begin and end? Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10907–10908. doi: 10.1073/pnas.90.23.10907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schumacher T. N., Mayr L. M., Minor D. L., Jr, Milhollen M. A., Burgess M. W., Kim P. S. Identification of D-peptide ligands through mirror-image phage display. Science. 1996 Mar 29;271(5257):1854–1857. doi: 10.1126/science.271.5257.1854. [DOI] [PubMed] [Google Scholar]
  35. Song L., Hobaugh M. R., Shustak C., Cheley S., Bayley H., Gouaux J. E. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science. 1996 Dec 13;274(5294):1859–1866. doi: 10.1126/science.274.5294.1859. [DOI] [PubMed] [Google Scholar]
  36. Valeva A., Weisser A., Walker B., Kehoe M., Bayley H., Bhakdi S., Palmer M. Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin. EMBO J. 1996 Apr 15;15(8):1857–1864. [PMC free article] [PubMed] [Google Scholar]
  37. Wade D., Boman A., Wåhlin B., Drain C. M., Andreu D., Boman H. G., Merrifield R. B. All-D amino acid-containing channel-forming antibiotic peptides. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4761–4765. doi: 10.1073/pnas.87.12.4761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Walker B., Bayley H. A pore-forming protein with a protease-activated trigger. Protein Eng. 1994 Jan;7(1):91–97. doi: 10.1093/protein/7.1.91. [DOI] [PubMed] [Google Scholar]
  39. Walker B., Bayley H. Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J Biol Chem. 1995 Sep 29;270(39):23065–23071. doi: 10.1074/jbc.270.39.23065. [DOI] [PubMed] [Google Scholar]
  40. 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. 1995 Feb;2(2):99–105. doi: 10.1016/1074-5521(95)90282-1. [DOI] [PubMed] [Google Scholar]
  41. Walker B., Krishnasastry M., Zorn L., Kasianowicz J., Bayley H. Functional expression of the alpha-hemolysin of Staphylococcus aureus in intact Escherichia coli and in cell lysates. Deletion of five C-terminal amino acids selectively impairs hemolytic activity. J Biol Chem. 1992 May 25;267(15):10902–10909. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES