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Fluorescence Spectroscopy

  • Catherine A. Royer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 40)

Abstract

The intrinsic fluorescence of aromatic amino acids in proteins has long been used as a means of monitoring unfolding/refolding transitions induced by chemical denaturants, temperature, pH changes, and pressure. The fluorescence properties of tryptophan residues in particular are exquisitely sensitive to perturbations of protein structure, whereas the low quantum yields of phenylalanine and tyrosine render these probes somewhat less useful for such studies. Accordingly, the following discussion of the application of fluorescence techniques to the study of protein folding will be limited to the intrinsic fluorescence of tryptophan in proteins and a few examples of the use of extrinsic fluorescent dyes. Although changes in fluorescence properties have been widely used to construct equilibrium and kinetic protein folding and unfolding profiles, rarely is there a detailed physical interpretation of these changes. Generally, protein folding is well described by a two-state process, and the profiles obtained monitoring fluorescence are coincident with those obtained by circular dichroism. In these cases, the fluorescence profiles are taken as indicators of general loss of protein structure, even though they arise from changes that are local to the tryptophan residue(s). In a number of studies, however, the fluorescence unfolding/refolding profiles, because they arise from structural changes local to particular tryptophan residues, have revealed non-two-state behavior (1, 2, 3). Thus, in some cases, fluorescence profiles can yield information about transient and stable intermediates in the folding/unfolding transition.

Keywords

Tryptophan Residue Rotational Correlation Time Denature State Folding Intermediate Denaturant Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Garvey, E. P. and Matthews, C. R. (1989) Biochemistry 28, 2083.PubMedCrossRefGoogle Scholar
  2. 2.
    Banik, U., Saha, R., Mandal, N. C, Bhattacharyya, B, and Roy, S. (1992) Eur. J Biochem. 206, 15–21.PubMedCrossRefGoogle Scholar
  3. 3.
    Mann, C. J., Royer, C. A., and Matthews, C. R. (1993) Prot. Sci. 2, 1853–1861.CrossRefGoogle Scholar
  4. 4.
    Szabo, A. G., Stepanik, T. M., Wayner, D. M., and Young, N. M. (1985) Biophys J. 41, 233–244.CrossRefGoogle Scholar
  5. 5.
    James, D. R., Demmer, D. R., Steer, R. P., and Verrall, R. E (1985) Biochemistry 24, 5517–5526.PubMedCrossRefGoogle Scholar
  6. 6.
    Beechem, J. M. and Brand, L. (1985) Ann. Rev. Biochem. 54, 43–71.PubMedCrossRefGoogle Scholar
  7. 7.
    Harris, D. L. and Hudson, B. S. (1990) Biochemistry 29, 5275–5284.Google Scholar
  8. 8.
    Eftink, M. R. (1991) Protein Structure Determination: Methods of Biochemical Analysis, vol. 35 (Suelter, C. H., ed.), Wiley, New York, pp 127–205.Google Scholar
  9. 9.
    Royer, C. A., Gardner, J. A., Beechem, J. M., Brochon, J.-C, and Matthews, K. S. (1990) Biophys. J. 58, 363–378.PubMedCrossRefGoogle Scholar
  10. 10.
    Axelson, P. H., Bajzer, Z, Prendergast, F. G., Cottam, P F., and Ho, C. (1991) Biophys. J. 60, 650–659.CrossRefGoogle Scholar
  11. 11.
    Royer, C. A. (1992) Biophys. J. 63, 741–750.PubMedCrossRefGoogle Scholar
  12. 12.
    Kim, S.-J., Chowdhury, F. H., Stryjewski, W., Younathan, E. S., Russo, P., and Barkley, M. D. (1993) Biophys. J. 65, 215–226.PubMedCrossRefGoogle Scholar
  13. 13.
    Kronman, M. J. and Holmes, L. G. (1971) Photochem. Photobiol. 14, 113–134.CrossRefGoogle Scholar
  14. 14.
    Grinvald, A. and Steinberg, I. Z. (1976) Biochim. Biophys. Acta 427, 663–678.PubMedGoogle Scholar
  15. 15.
    Janes, S. M., Holtom, G., Ascenzi, P., Brunori, M., and Hochstrasser, R. M. (1987) Biophys. J. 51, 653–660.PubMedCrossRefGoogle Scholar
  16. 16.
    Gryczynski, I., Eftink, M., and Lakowicz, J. R. (1988) Biochim. Biophys. Acta 954, 244–252.PubMedCrossRefGoogle Scholar
  17. 17.
    Eftink, M. R., Gryczynski, I., Wiczk, W., Laczko, G., and Lakowicz, J. R. (1991) Biochemistry 30, 8945–8953.PubMedCrossRefGoogle Scholar
  18. 18.
    Perrin, F. (1926) J. Phys. Radium 1, 390–401.CrossRefGoogle Scholar
  19. 19.
    Royer, C. A., Mann, C. J., and Matthews, C. R. (1993) Prot. Sci. 2, 1844–1852.CrossRefGoogle Scholar
  20. 20.
    Fernando, T. and Royer, C. A (1992) Biochemistry 31, 6683–6691PubMedCrossRefGoogle Scholar
  21. 21.
    Lane, A. N. and Jardetsky, O (1987) Eur. J. Biochem. 164, 389–396.PubMedCrossRefGoogle Scholar
  22. 22.
    Perez, G. M., Howard, P., Weil, A., and Beechem, J. M. (1992) Biophys. J. 61, 2805.Google Scholar
  23. 23.
    Labhardt, A. M. (1986) Methods Enzymol. 131, 126–135.PubMedCrossRefGoogle Scholar
  24. 24.
    Kuwajima, K., Garvey, E. P., Finn, B. E., Matthews, C. R., and Sugai, S. (1991) Biochemistry 30, 7693–7703.PubMedCrossRefGoogle Scholar
  25. 25.
    Goldberg, M. E., Semisotnov, G. V., Friguet, B., Kuwajima, K., Ptitsyn, O. B., and Sugai, S. (1990) FEBS Lett. 263, 51–56.PubMedCrossRefGoogle Scholar
  26. 26.
    Seethaler, N. and Beechem, J. M. (1992) Biophys. J. 61, A179.Google Scholar
  27. 27.
    Beechem, J. M. (1992) S.P.I.E. Proceedings 1640: Time-Resolved Laser Spectroscopy in Biochemistry III, 676–680.Google Scholar
  28. 28.
    Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991) Bopolymers 31, 119–128.CrossRefGoogle Scholar
  29. 29.
    Beechem, J. M., Gratton, E., Ameloot, M. A., Knutson, J. R., and Brand, L. (1991) in Topics in Fluorescence Spectroscopy, vol. 2 (Lakowicz, J. R., ed.), Plenum, New York, pp. 241–301.Google Scholar
  30. 30.
    Royer, C. A., Hinck, A. P., Loh, S. N., Prehoda, K. E., Peng, X., Jonas, J., and Markley, J. L. (1993) Biochemistry 32, 5222–5232.PubMedCrossRefGoogle Scholar
  31. 31.
    Amir, D., Levy, D. P., Levin, Y., and Haas, E. (1986) Biopolymers 25, 1645–1658.PubMedCrossRefGoogle Scholar
  32. 32.
    Amir, D. and Haas, E. (1987) Biochemistry 26, 2162–2175.PubMedCrossRefGoogle Scholar
  33. 33.
    Amir, D. and Haas, E. (1988) Biochemistry 27, 8889–8893.PubMedCrossRefGoogle Scholar
  34. 34.
    James, E., Wu, P. G., Stites, W., and Brand, L. (1992) Biochemistry 31, 10, 217–10,225.Google Scholar
  35. 35.
    Walbridge, D., Knutson, J. R., and Brand, L. (1987) Anal. Biochem. 161, 167–178.CrossRefGoogle Scholar
  36. 36.
    Eriksson, S., Tetm, S., Voss, E., Gratton, E., and Mantulin, W. (1993) Biophys. J. 61, A218.Google Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Catherine A. Royer
    • 1
  1. 1.School of PharmacyUniversity of WisconsinMadisonWI

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