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. 2000 Oct;79(4):1967–1975. doi: 10.1016/S0006-3495(00)76445-9

Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore.

L Q Gu 1, H Bayley 1
PMCID: PMC1301087  PMID: 11023901

Abstract

Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alphaHL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a biosensor element. To further such studies, it is important to find conditions under which the dwell time of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (betaCD) with alphaHL. betaCD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for betaCD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral betaCD molecule varies with pH and voltage, a result that is incompatible with two states of the alphaHL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alphaHL. betaCD complex varies continuously with the transmembrane potential.

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Selected References

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

  1. Bayley H. Building doors into cells. Sci Am. 1997 Sep;277(3):62–67. doi: 10.1038/scientificamerican0997-62. [DOI] [PubMed] [Google Scholar]
  2. Bayley H. Designed membrane channels and pores. Curr Opin Biotechnol. 1999 Feb;10(1):94–103. doi: 10.1016/s0958-1669(99)80017-2. [DOI] [PubMed] [Google Scholar]
  3. Bezrukov S. M. Ion channels as molecular coulter counters to probe metabolite transport. J Membr Biol. 2000 Mar 1;174(1):1–13. doi: 10.1007/s002320001026. [DOI] [PubMed] [Google Scholar]
  4. Bezrukov S. M., Kasianowicz J. J. The charge state of an ion channel controls neutral polymer entry into its pore. Eur Biophys J. 1997;26(6):471–476. doi: 10.1007/s002490050101. [DOI] [PubMed] [Google Scholar]
  5. Bezrukov SM, Kasianowicz JJ. Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel. Phys Rev Lett. 1993 Apr 12;70(15):2352–2355. doi: 10.1103/PhysRevLett.70.2352. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Botsi A., Yannakopoulou K., Hadjoudis E., Waite J. AM1 calculations on inclusion complexes of cyclomaltoheptaose (beta-cyclodextrin) with 1,7-dioxaspiro[5.5]undecane and nonanal, and comparison with experimental results. Carbohydr Res. 1996 Mar 22;283:1–16. doi: 10.1016/0008-6215(95)00410-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. D'Souza Valerian T., Lipkowitz Kenny B. Cyclodextrins: Introduction. Chem Rev. 1998 Jul 30;98(5):1741–1742. doi: 10.1021/cr980027p. [DOI] [PubMed] [Google Scholar]
  11. Eroglu A., Russo M. J., Bieganski R., Fowler A., Cheley S., Bayley H., Toner M. Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol. 2000 Feb;18(2):163–167. doi: 10.1038/72608. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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. 1999 Apr 22;398(6729):686–690. doi: 10.1038/19491. [DOI] [PubMed] [Google Scholar]
  15. Gu L. Q., Dalla Serra M., Vincent J. B., Vigh G., Cheley S., Braha O., Bayley H. Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters. Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):3959–3964. doi: 10.1073/pnas.97.8.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kasianowicz J. J., Bezrukov S. M. Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations. Biophys J. 1995 Jul;69(1):94–105. doi: 10.1016/S0006-3495(95)79879-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Korchev Y. E., Alder G. M., Bakhramov A., Bashford C. L., Joomun B. S., Sviderskaya E. V., Usherwood P. N., Pasternak C. A. Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells. J Membr Biol. 1995 Jan;143(2):143–151. doi: 10.1007/BF00234660. [DOI] [PubMed] [Google Scholar]
  19. Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
  20. Krasilnikov O. V., Capistrano M. P., Yuldasheva L. N., Nogueira R. A. Influence of Cys-130 S. aureus alpha-toxin on planar lipid bilayer and erythrocyte membranes. J Membr Biol. 1997 Mar 15;156(2):157–172. doi: 10.1007/s002329900198. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Merzlyak P. G., Yuldasheva L. N., Rodrigues C. G., Carneiro C. M., Krasilnikov O. V., Bezrukov S. M. Polymeric nonelectrolytes to probe pore geometry: application to the alpha-toxin transmembrane channel. Biophys J. 1999 Dec;77(6):3023–3033. doi: 10.1016/S0006-3495(99)77133-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Pederzolli C., Belmonte G., Dalla Serra M., Macek P., Menestrina G. Biochemical and cytotoxic properties of conjugates of transferrin with equinatoxin II, a cytolysin from a sea anemone. Bioconjug Chem. 1995 Mar-Apr;6(2):166–173. doi: 10.1021/bc00032a003. [DOI] [PubMed] [Google Scholar]
  26. Saenger Wolfram, Jacob Joël, Gessler Katrin, Steiner Thomas, Hoffmann Daniel, Sanbe Haruyo, Koizumi Kyoko, Smith Steven M., Takaha Takeshi. Structures of the Common Cyclodextrins and Their Larger Analogues-Beyond the Doughnut. Chem Rev. 1998 Jul 30;98(5):1787–1802. doi: 10.1021/cr9700181. [DOI] [PubMed] [Google Scholar]
  27. Schneider Hans-Jörg, Hacket Frank, Rüdiger Volker, Ikeda Hiroshi. NMR Studies of Cyclodextrins and Cyclodextrin Complexes. Chem Rev. 1998 Jul 30;98(5):1755–1786. doi: 10.1021/cr970019t. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Szejtli József. Introduction and General Overview of Cyclodextrin Chemistry. Chem Rev. 1998 Jul 30;98(5):1743–1754. doi: 10.1021/cr970022c. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ziegler C., Göpel W. Biosensor development. Curr Opin Chem Biol. 1998 Oct;2(5):585–591. doi: 10.1016/s1367-5931(98)80087-2. [DOI] [PubMed] [Google Scholar]
  34. al-yahyaee S. A., Ellar D. J. Cell targeting of a pore-forming toxin, CytA delta-endotoxin from Bacillus thuringiensis subspecies israelensis, by conjugating CytA with anti-Thy 1 monoclonal antibodies and insulin. Bioconjug Chem. 1996 Jul-Aug;7(4):451–460. doi: 10.1021/bc960030k. [DOI] [PubMed] [Google Scholar]

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