• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. Jun 1999; 8(6): 1257–1267.
PMCID: PMC2144353

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

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 (2.4M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Braha O, Walker B, Cheley S, Kasianowicz JJ, Song L, Gouaux JE, Bayley H. Designed protein pores as components for biosensors. Chem Biol. 1997 Jul;4(7):497–505. [PubMed]
  • Chang CY, Niblack B, Walker B, Bayley H. A photogenerated pore-forming protein. Chem Biol. 1995 Jun;2(6):391–400. [PubMed]
  • Cheley S, Malghani MS, Song L, Hobaugh M, Gouaux JE, 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. [PubMed]
  • Chorev M, Goodman M. Recent developments in retro peptides and proteins--an ongoing topochemical exploration. Trends Biotechnol. 1995 Oct;13(10):438–445. [PubMed]
  • 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. [PubMed]
  • Füssle R, Bhakdi S, Sziegoleit A, Tranum-Jensen J, Kranz T, Wellensiek HJ. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. [PMC free article] [PubMed]
  • Gouaux E. alpha-Hemolysin from Staphylococcus aureus: an archetype of beta-barrel, channel-forming toxins. J Struct Biol. 1998;121(2):110–122. [PubMed]
  • Gouaux JE, Braha O, Hobaugh MR, 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. [PMC free article] [PubMed]
  • Guptasarma P. Reversal of peptide backbone direction may result in the mirroring of protein structure. FEBS Lett. 1992 Oct 5;310(3):205–210. [PubMed]
  • Heinz DW, Baase WA, Matthews BW. 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. [PMC free article] [PubMed]
  • Hutchinson EG, Sessions RB, Thornton JM, Woolfson DN. Determinants of strand register in antiparallel beta-sheets of proteins. Protein Sci. 1998 Nov;7(11):2287–2300. [PMC free article] [PubMed]
  • Ido Y, Vindigni A, Chang K, Stramm L, Chance R, Heath WF, DiMarchi RD, Di Cera E, Williamson JR. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science. 1997 Jul 25;277(5325):563–566. [PubMed]
  • Jones DH, Howard BH. 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]
  • Kasianowicz JJ, Brandin E, Branton D, Deamer DW. Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13770–13773. [PMC free article] [PubMed]
  • Korchev YE, Bashford CL, Alder GM, Kasianowicz JJ, Pasternak CA. Low conductance states of a single ion channel are not 'closed'. J Membr Biol. 1995 Oct;147(3):233–239. [PubMed]
  • Krasilnikov OV, Sabirov RZ, Ternovsky VI, Merzliak PG, Muratkhodjaev JN. 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. [PubMed]
  • Krasilnikov OV, Sabirov RZ, Ternovsky VI, Merzliak PG, Tashmukhamedov BA. The structure of Staphylococcus aureus alpha-toxin-induced ionic channel. Gen Physiol Biophys. 1988 Oct;7(5):467–473. [PubMed]
  • Lacroix E, Viguera AR, Serrano L. Reading protein sequences backwards. Fold Des. 1998;3(2):79–85. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • MacKenzie KR, Engelman DM. 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. [PMC free article] [PubMed]
  • Matthews BW, Weaver LH, Kester WR. The conformation of thermolysin. J Biol Chem. 1974 Dec 25;249(24):8030–8044. [PubMed]
  • 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. [PubMed]
  • Merrifield RB, Juvvadi P, Andreu D, Ubach J, Boman A, Boman HG. Retro and retroenantio analogs of cecropin-melittin hybrids. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3449–3453. [PMC free article] [PubMed]
  • Middaugh CR, Thomson JA, Burke CJ, Mach H, Naylor AM, Bogusky MJ, Ryan JA, Pitzenberger SM, Ji H, Cordingley JS. Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni. Protein Sci. 1993 Jun;2(6):900–914. [PMC free article] [PubMed]
  • Milton RC, Milton SC, Kent SB. 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Nicholson H, Söderlind E, Tronrud DE, Matthews BW. 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. [PubMed]
  • 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. [PubMed]
  • Olszewski KA, Kolinski A, Skolnick J. Does a backwardly read protein sequence have a unique native state? Protein Eng. 1996 Jan;9(1):5–14. [PubMed]
  • Panchal RG, Cusack E, Cheley S, Bayley H. Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat Biotechnol. 1996 Jul;14(7):852–856. [PubMed]
  • Regan L. What determines where alpha-helices begin and end? Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10907–10908. [PMC free article] [PubMed]
  • Schumacher TN, Mayr LM, Minor DL, Jr, Milhollen MA, Burgess MW, Kim PS. Identification of D-peptide ligands through mirror-image phage display. Science. 1996 Mar 29;271(5257):1854–1857. [PubMed]
  • Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science. 1996 Dec 13;274(5294):1859–1866. [PubMed]
  • 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]
  • Wade D, Boman A, Wåhlin B, Drain CM, Andreu D, Boman HG, Merrifield RB. All-D amino acid-containing channel-forming antibiotic peptides. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4761–4765. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PubMed]
  • Walker B, Bayley H. A pore-forming protein with a protease-activated trigger. Protein Eng. 1994 Jan;7(1):91–97. [PubMed]
  • 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]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...