Skip to main content
SpringerLink
Log in

Pore-forming protein structure analysis in membranes using multiple independent fluorescence techniques

  • Review Article
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

A large number of transmembrane proteins form aqueous pores or channels in the phospholipid bilayer, but the structural bases of pore formation and assembly have been determined experimentally for only a few of the proteins and protein complexes. The polypeptide segments that form the transmembrane pore and the secondary structure that creates the aqueous-lipid interface can be identified using multiple independent fluorescence techniques (MIFT). The information obtained from several different, but complementary, fluorescence analyses, including measurements of emission intensity, fluorescence lifetime, accessibility to aqueous and to lipophilic quenching agents, and fluorescence resonance energy transfer (FRET) can be combined to characterize the nature of the protein-membrane interaction directly and unambiguously. The assembly pathway can also be determined by measuring the kinetics of the spectral changes that occur upon pore formation. The MIFT approach therefore allows one to obtain structural information that cannot be obtained easily using alternative techniques such as crystallography. This review briefly outlines how MIFT can reveal the identity, location, conformation, and topography of the polypeptide sequences that interact with the membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Schirmer, T. (1998) General and specific porins from bacterial outer membranes. J. Struct. Biol. 121, 101–109.

    Article  PubMed  CAS  Google Scholar 

  2. Johnson, A. E. and van Waes, M. A. (1999) The translocon: a dynamic gateway at the ER membrane. Annu. Rev. Cell Dev. Biol. 15, 799–842.

    Article  PubMed  Google Scholar 

  3. Rassow, J. and Pfanner, N. (2000) The protein import machinery of the mitochondrial membranes. Traffic 1, 457–464.

    Article  PubMed  CAS  Google Scholar 

  4. Alouf, J. E. and Freer, J. H. (1999) The Comprehensive Sourcebook of Bacterial Protein Toxins, 2nd ed., Academic, London.

    Google Scholar 

  5. Heuck, A. P., Tweten, R. K., and Johnson, A. E. (2001) β-Barrel pore forming toxins: intriguing dimorphic proteins. Biochemistry 40, 9065–9073.

    Article  PubMed  CAS  Google Scholar 

  6. Lakey, J. H. and Slatin, S. L. (2001) Pore-forming colicins and their relatives, in Pore-Forming Toxins (van der Goot, F. G., ed.), Current Topics in Microbiology and Immunology Series, Springer-Verlag, Berlin, pp. 131–161.

    Google Scholar 

  7. Hamman, B. D., Chen, J.-C., Johnson, E. E., and Johnson, A. E. (1997) The aqueous pore through the translocon has a diameter of 40–60 Å during cotranslational protein translocation at the ER membrane. Cell 89, 535–544.

    Article  PubMed  CAS  Google Scholar 

  8. Hamman, B. D., Hendershot, L. M., and Johnson, A. E. (1998) BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92, 747–758.

    Article  PubMed  CAS  Google Scholar 

  9. Gabriel, K., Buchanan, S. K., and Lithgow, T. (2001) The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes. Trends Biochem. Sci. 26, 36–40.

    Article  PubMed  CAS  Google Scholar 

  10. White, S. H. and Wimley, W. C. (1999) Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28, 319–365.

    Article  PubMed  CAS  Google Scholar 

  11. Baleja, J. D. (2001) Structure determination of membrane-associated proteins from nuclear magnetic resonance data. Anal. Biochem. 288, 1–15.

    Article  PubMed  CAS  Google Scholar 

  12. White, S. H., Ladokhin, A. S., Jayasinghe, S., and Hristova, K. (2001) How membranes shape protein structure. J. Biol. Chem. 276, 32,395–32,398.

    CAS  Google Scholar 

  13. Hubbell, W. L., Gross, A., Langen, R., and Lietzow, M. A. (1998) Recent advances in sitedirected spin labeling of proteins. Curr. Opin. Struct. Biol. 8, 649–656.

    Article  PubMed  CAS  Google Scholar 

  14. Vigano, C., Manciu, L., Buyse, F., Goormaghtigh, E. and Ruysschaert, J.-M. (2000) Attenuated total reflection IR spectroscopy as a tool to investigate the structure, orientation and tertiary structure changes in peptides and membrane proteins. Biopolymers 55, 373–380.

    Article  PubMed  CAS  Google Scholar 

  15. Chen, Y. and Barkley, M. D. (1998) Toward understanding tryptophan fluorescence in proteins. Biochemistry 37, 9976–9982.

    Article  PubMed  CAS  Google Scholar 

  16. Johnson, A. E., Esmon, N. L., Laue, T. M., and Esmon, C. T. (1983) Structural changes required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain γ-carboxyglutamic acid. J. Biol. Chem. 258, 5554–5560.

    PubMed  CAS  Google Scholar 

  17. Soulages, J. L. and Arrese, E. L. (2000) Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipopoproteins of dimyristoylphosphatidylcholine. Biochemistry 39, 10,574–10,580.

    Article  CAS  Google Scholar 

  18. Nakamura, M., Sekino-Suzuki, N., Mitsui, K.-I., and Ohno-Iwashita, Y. (1998) Contribution of tryptophan residues to the structural changes in perfringolysin O during interaction with liposomal membranes J. Biochem. 123, 1145–1155.

    PubMed  CAS  Google Scholar 

  19. Shepard, L. A., Heuck, A. P., Hamman, B. D., Rossjohn, J., Parker, M. W., Ryan, K. R., et al. (1998) Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an α-helical to β-sheet transition identified by fluorescence spectroscopy. Biochemistry 37, 14,563–14,574.

    Article  CAS  Google Scholar 

  20. Ye, J., Esmon, N. L., Esmon, C. T., and Johnson, A. E. (1991) The active site of thrombin is altered upon binding to thrombomodulin: two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. J. Biol. Chem. 266, 23,016–23,021.

    CAS  Google Scholar 

  21. Hotze, E. M., Wilson-Kubalek, E. M., Rossjohn, J., Parker, M. W., Johnson, A. E. and Tweten, R. K. (2001) Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane β-sheet from a prepore intermediate. J. Biol. Chem. 276, 8261–8268.

    Article  PubMed  CAS  Google Scholar 

  22. Arai, K.-I., Kawakita, M., Nakamura, S., Ishikawa, I. and Kaziro, Y. (1974) Studies on the polypeptide elongation factors from E. coli. IV. Characterization of sulfhydryl groups in EF-Tu and EF-Ts. J. Biochem. (Tokyo) 76, 523–534.

    CAS  Google Scholar 

  23. Mansoor, S. E., Mchaourab, H. S. and Farrens, D. L. (1999) Determination of protein secondary structure and solvent accessibility using sitedirected fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane. Biochemistry 38, 16,383–16,393.

    Article  CAS  Google Scholar 

  24. Crowley, K. S., Liao, S., Worrell, V. E., Reinhart, G. D., and Johnson, A. E. (1994) Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461–471.

    Article  PubMed  CAS  Google Scholar 

  25. Liao, S., Lin, J., Do, H. and Johnson, A. E. (1997) Both lumenal and cytosolic gating of the aqueous ER translocon pore is regulated from inside the ribosome during membrane protein integration. Cell 90, 31–41.

    Article  PubMed  CAS  Google Scholar 

  26. Husten, E. J., Esmon, C. T. and Johnson, A. E. (1987) The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex. J. Biol. Chem. 262, 12,953–12,961.

    CAS  Google Scholar 

  27. Yegneswaran, S., Smirnov, M. D., Safa, O., Esmon, N. L., Esmon, C. T. and Johnson, A. E. (1999) Relocating the active site of activated protein C eliminates the need for its protein S cofactor. A fluorescence resonance energy transfer study. J. Biol. Chem. 274, 5462–5468.

    Article  PubMed  CAS  Google Scholar 

  28. Heuck, A. P., Hotze, E. M., Tweten, R. K., and Johnson, A. E. (2000) Mechanism of membrane insertion of a multimeric β-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes. Mol. Cell 6, 1233–1242.

    Article  PubMed  CAS  Google Scholar 

  29. MacDonald, R. C., MacDonald, R. I., Menco, B. P. M., Takeshita, K., Subbarao, N. K. and Hu, L. (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim. Biophys. Acta 1061 297–303.

    Article  PubMed  CAS  Google Scholar 

  30. Mayer, L. D., Hope, M. J. and Cullis, P. R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858, 161–168.

    Article  PubMed  CAS  Google Scholar 

  31. Weber, G. and Farris, F. J. (1979) Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry 18, 3075–3078.

    Article  PubMed  CAS  Google Scholar 

  32. Prendergast, F. G., Meyer, M., Carlson, G. L., Iida, S., and Potter, J. D. (1983) Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). A thiol-selective, polarity-sensitive fluorescent probe. J. Biol. Chem. 258, 7541–7544.

    PubMed  CAS  Google Scholar 

  33. Valeva, A., Weisser, A., Walker, B., Kehoe, M., Bayley, H., Bhakdi, S., et al. (1996) Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal α-toxin. EMBO J. 15, 1857–1864.

    PubMed  CAS  Google Scholar 

  34. Schindel, C., Zitzer, A., Schulte, B., Gerhards, A., Stanley, P., Hughes, C., et al. (2001) Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis. Eur. J. Biochem. 268, 800–808.

    Article  PubMed  CAS  Google Scholar 

  35. Kenner, R. A. and Aboderin, A. A. (1971) A new fluorescent probe for protein and nucleoprotein conformation. Binding of 7-(p-methoxybenzylamino)-4-nitrobenzoxadiazole to bovine trypsinogen and bacterial ribosomes. Biochemistry 10, 4433–4440.

    Article  PubMed  CAS  Google Scholar 

  36. Crowley, K., Reinhart, G. D. and Johnson, A. E. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115.

    Article  PubMed  CAS  Google Scholar 

  37. Kosower, E. M. and Kosower, N. S. (1995) Bromobimane probes for thiols. Methods Enzymol. 251, 133–148.

    PubMed  CAS  Google Scholar 

  38. Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J. and London, E. (1997) Identification of shallow and deep membranepenetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width. J. Biol. Chem. 272, 25,091–25,098.

    CAS  Google Scholar 

  39. Shatursky, O., Heuck, A. P., Shepard, L. A., Rossjohn, J., Parker, M. W., Johnson, A. E., et al. (1999) The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 99, 293–299.

    Article  PubMed  CAS  Google Scholar 

  40. Chattopadhyay, A. and London, E. (1987) Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26, 39–45.

    Article  PubMed  CAS  Google Scholar 

  41. Mutucumarana, V. P., Duffy, E. J., Lollar, P., and Johnson, A. E. (1992) The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study. J. Biol. Chem. 267, 17,012–17,021.

    CAS  Google Scholar 

  42. McCallum, C. D., Hapak, R. C., Neuenschwander, P. F., Morrissey, J. H., and Johnson, A. E. (1996) The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study. J. Biol. Chem. 271, 28,168–28,175.

    CAS  Google Scholar 

  43. Yegneswaran, S., Wood, G. M., Esmon, C. T., and Johnson, A. E. (1997) Protein S alters the active site location of activated protein C above the membrane surface. A fluorescence resonance energy transfer study of topography. J. Biol. Chem. 272, 25,013–25,021.

    Article  CAS  Google Scholar 

  44. Armstrong, S. A., Husten, E. J., Esmon, C. T., and Johnson, A. E. (1990) The active site of membrane-bound meizothrombin: a fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va. J. Biol. Chem. 265, 6210–6218.

    PubMed  CAS  Google Scholar 

  45. Lu, R., Esmon, N. L., Esmon, C. T. and Johnson, A. E. (1989) The active site of the thrombin-thrombomodulin complex: a fluorescence energy transfer measurement of its distance above the membrane surface. J. Biol. Chem. 264, 12,956–12,962.

    CAS  Google Scholar 

  46. Duffy, E. J., Parker, E. T., Mutucumarana, V. P., Johnson, A. E. and Lollar, P. (1992) Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles. J. Biol. Chem. 267, 17,006–17,011.

    CAS  Google Scholar 

  47. Harris, R. W., Sims, P. J. and Tweten, R. K. (1991) Kinetic aspects of the aggregation of Clostridium perfringens θ-toxin on erythrocyte membranes. A fluorescence energy transfer study. J. Biol. Chem. 266, 6936–6941.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, 77843-1114, College Station, TX

    Alejandro P. Heuck & Arthur E. Johnson

  2. Departments of Chemistry, and of Biochemistry and Biophysics, Texas A&M University, 77843, College Station, TX

    Arthur E. Johnson

Authors
  1. Alejandro P. Heuck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arthur E. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Arthur E. Johnson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Heuck, A.P., Johnson, A.E. Pore-forming protein structure analysis in membranes using multiple independent fluorescence techniques. Cell Biochem Biophys 36, 89–101 (2002). https://doi.org/10.1385/CBB:36:1:89

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/CBB:36:1:89

Index Entries

Search

Navigation