Journal of Protein Chemistry

, Volume 15, Issue 2, pp 205–218 | Cite as

A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study

  • Jean-Alain Bousquet
  • Norbert Ettner


A thorough investigation of the acrylamide fluorescence quenching of F75TetR, a mutant of the Tn10-encoded TetR repressor containing a single Trp residue at position 43, was carried out. The Trp-43 residue is located in a helixα-turn-helixα (H-t-H) motif involved in the specific binding of F75TetR to the operator site in specific DNA. Distinct Ranges of acrylamide concentration have been assumed. At acrylamide concentrations below 0.15–0.2 M (a usual range of values in fluorescence quenching studies) the observed limited tertiary structure change induced by acrylamide is consistent with a noncooperative local unfolding of the DNA-binding domain. It is suggested that penetration of the neutral quencher could cause the deletion of a hydrophobic tertiary structure contact, partly involving TrP-43, responsible for the anchoring of the H-t-H motif inside the three-helix protein bundle, characterizing the N-terminal part. Correspondingly, the affinity of the mutant repressor for the operator was shown to decrease substantially (about five orders of magnitude), seemingly losing its specificity. A subsequent phase, up to 0.8 M acrylamide, was observed in which the involved intermediate protein structure is not further perturbed, nor is DNA binding.

Key words

Acrylamide fluorescence quenching Tet repressor H-t-H motif protein-DNA binding 







engineered tetracycline repressor in which the Trp residue at the position 75 in the wild-type repressor TetR is replaced by a Phe residue


helixα-turn-helixα super-secondary structure


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  1. Altschmied, L., Baumeister, R., Pleiderer, K., and Hillen, W. (1988).EMBO J. 7, 4011–4017.PubMedGoogle Scholar
  2. Baumeister, R., Muller, G., Hecht, B., and Hillen, W. (1992).Proteins 14, 168–177.PubMedGoogle Scholar
  3. Beck, C. G., Mutzel, R., Barbe, J., and Muller, W. (1982).J. Bacteriol. 150, 633–642.PubMedGoogle Scholar
  4. Berens, C., Altshmied, L., and Hillen, W. (1992).J. Biol. Chem. 267, 1945–1952.PubMedGoogle Scholar
  5. Bertrand, K. P., Postle, K., Wray, L. V., and Reznikoff, W. S. (1983).Gene 23, 149–156.PubMedGoogle Scholar
  6. Blatt, E., Husain, A., and Sawyer, W. H. (1986).Biochim. Biophys. Acta 871, 6–13.PubMedGoogle Scholar
  7. Boczko, E. M., and Brooks, C. L. (1995).Science 269, 393–396.PubMedGoogle Scholar
  8. Brennan, R. G., and Matthews, B. W. (1989).J. Biol. Chem. 264, 1903–1906.PubMedGoogle Scholar
  9. Calhoun, D. B., Vanderkooi, J. M., and Englander, S. W. (1983).Biochemistry 22, 1533–1539.PubMedGoogle Scholar
  10. Carell, R. W., Evans, O., and Stein, P. E. (1991).Nature 353, 576–578.PubMedGoogle Scholar
  11. Carell, R. W., Evans, O., and Stein, P. E. (1993).Nature 364, 737.Google Scholar
  12. Cavins, J. F., and Friedman, M. (1968).J. Biol. Chem. 243, 3357.PubMedGoogle Scholar
  13. Chabbert, M., Hillen, W., Hansen, D., Takahashi, M., and Bousquet, J. A. (1992).Biochemistry 31, 1951–1960.PubMedGoogle Scholar
  14. Danilveiante, M., Adomeniene, O., and Dienys, G. (1981).Org. Reactiv. 18, 217–224.Google Scholar
  15. Dodd, I. B., and Eagan, J. B. (1987).J. Mol. Biol. 194, 557–564.PubMedGoogle Scholar
  16. Eftink, M. R., and Ghiron, C. A. (1975).Proc. Natl. Acad. Sci. USA 72, 3290–3294.PubMedGoogle Scholar
  17. Eftink, M. R., and Ghiron, C. A. (1976).Biochemistry 15, 672–680.PubMedGoogle Scholar
  18. Eftink, M. R., and Ghiron, C. A. (1977).Biochemistry 16, 5546–5551.PubMedGoogle Scholar
  19. Eftink, M. R., and Ghiron, C. A. (1981).Anal. Biochem. 114, 199–227.CrossRefPubMedGoogle Scholar
  20. Eftink, M. R., and Ghiron, C. A. (1984).Biochemistry 23, 6891–6899.Google Scholar
  21. Eftink, M. R., and Ghiron, C. A. (1987).Biochem. Biophys. Acta 916, 343–349.PubMedGoogle Scholar
  22. Eftink, M. R., and Selvidge, L. A. (1982).Biochemistry 21, 117–125.PubMedGoogle Scholar
  23. Eftink, M. R., Wasylewsky, Z., and Ghiron, C. A. (1987).Biochemistry 26, 8338–8346.PubMedGoogle Scholar
  24. Englander, S. W. (1975).Ann. N. Y. Acad. Sci. 244, 10–27.PubMedGoogle Scholar
  25. Englander, S. W., and Kallenbach, N. R. (1983).Q. Rev. Biophys. 16, 521–655.PubMedGoogle Scholar
  26. Follenius, A., and Gerard, D. (1983).Photochem. Photobiol. 38, 373–376.PubMedGoogle Scholar
  27. Geisthardt, D., and Kruppa, J. (1987).Anal. Biochem. 160, 184–191.PubMedGoogle Scholar
  28. Gratton, E., Jameson, D. M., Weber, G., and Alpert, B. (1984).Biophys. J. 45, 789–794.PubMedGoogle Scholar
  29. Hagaman, K. A., and Eftink, M. R. (1984).Biophys. Chem. 20, 201–207.PubMedGoogle Scholar
  30. Hansen, D., and Hillen, W. (1987).J. Biol. Chem. 262, 12269–12274.PubMedGoogle Scholar
  31. Hansen, D., Altshmied, L., and Hillen, W. (1987).J. Biol. Chem. 262, 14030–14035.PubMedGoogle Scholar
  32. Harrison, S. C., and Aggarval, A. K. (1990).Annu. Rev. Biochem. 59, 933–969.PubMedGoogle Scholar
  33. Haynie, D. T., and Freire, E. (1993).Proteins Struct. Funct. Genet. 16, 115–140.PubMedGoogle Scholar
  34. Hillen, W., and Berens, C. (1994).Annu. Rev. Microbiol. 48, 345–369.PubMedGoogle Scholar
  35. Hillen, W., and Schollmeier, K. (1983).Nucleic Acids Res. 11, 525–539.PubMedGoogle Scholar
  36. Hillen, W., and Unger, B. (1982).Nature 297, 700–702.PubMedGoogle Scholar
  37. Hillen, W., Klock, G., Kaffenberger, I., Wray, L. V., and Reznikoff, W. S. (1982).J. Biol. Chem. 257, 6605–6613.PubMedGoogle Scholar
  38. Hillen, W., Gatz, C., Altshmied, L., Schollmeier, K., and Meier, I. (1983).J. Mol. Biol. 169, 707–721.PubMedGoogle Scholar
  39. Hillen, W., Schollmeier, K., and Gatz, C. (1984).J. Mol. Biol. 172, 185–201.PubMedGoogle Scholar
  40. Hinrichs, W., Kisker, C., Düvel, M., Müller, A., Tovar, K., Hillen, W., and Saenger, W. (1994).Science 264, 418–420.PubMedGoogle Scholar
  41. Ilich, P., and Prendergast, F. G. (1991).Photochem. Photobiol. 53, 445–453.PubMedGoogle Scholar
  42. Isackson, P. J., and Bertrand, K. P. (1985).Proc. Natl. Acad. Sci. USA 82, 6226–6230.PubMedGoogle Scholar
  43. Jameson, D. M., Gratton, E., Weber, G., and Alpert, B. (1984).Biophys. J. 45, 795–803.PubMedGoogle Scholar
  44. Jordan, S. R., and Pabo, C. O. (1988).Science 242, 893–899.PubMedGoogle Scholar
  45. Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., and Saenger, W. (1995).J. Mol. Biol. 247, 260–280.PubMedGoogle Scholar
  46. Kleinschimdt, C., Tovar, K., Hillen, W., and Porschke, D. (1988).Biochemistry 27, 1094–1104.PubMedGoogle Scholar
  47. Kleinschimdt, C., Tovar, K., and Hillen, W. (1991).Nucleic Acids Res. 19, 1021–1028.PubMedGoogle Scholar
  48. Kulus, J. (1981).Biotechnol. Bioeng. 23, 2875.Google Scholar
  49. Kurban, G. P., Gitlin, G., Bayer, E. A., Walchek, M., and Horowitz, P. M. (1990).J. Protein Chem. 9, 673–682.PubMedGoogle Scholar
  50. Kuwajima, K. (1989).Proteins Struct. Funct. Genet. 6, 87–103.PubMedGoogle Scholar
  51. Lakowicz, J. R. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York, pp. 111–153.Google Scholar
  52. Lakowicz, J. R., and Weber, G. (1973).Biochemistry 12, 4161–4171.PubMedGoogle Scholar
  53. Lakowicz, J. R., and Weber, G. (1973).Biochemistry 53, 445–453.Google Scholar
  54. Lederer, H., Tovar, K., Baer, G., May, R. P., Hillen, W., and Heumann, H. (1989).EMBO J. 8, 1257–1263.PubMedGoogle Scholar
  55. Matko, J., Tron, L., Balazs, M., Hevessy, J., Somogyi, B., and Damjanovich, S. (1980).Biochemistry 19, 5782–5786.PubMedGoogle Scholar
  56. McGhee, J. D., and von Hippel, P. H. (1974).J. Mol. Biol. 86, 469–489.PubMedGoogle Scholar
  57. Meier, I., Wray, L. W., and Hillen, W. (1988).EMBO J. 7, 567–572.PubMedGoogle Scholar
  58. Merill, A. R., Palmer, L. R., and Szabo, A. G. (1993).Biochemistry 32, 6974–6981.PubMedGoogle Scholar
  59. Mondragon, A., and Harrison, S. C. (1991).J. Mol. Biol. 219, 321–334.PubMedGoogle Scholar
  60. Narasimhulu, S. (1991).Biochemistry 30, 9319–9327.PubMedGoogle Scholar
  61. Ngo, T. T. (1976).Int. J. Biochem. 7, 193–197.Google Scholar
  62. Niederweis, M., and Hillen, W. (1993).Electrophoresis 14, 693–698.PubMedGoogle Scholar
  63. Nowak, M. W., and Berman, H. A. (1991).Biochemistry 30, 7642–7651.PubMedGoogle Scholar
  64. Pitsyn, O. B. (1987).J. Protein Chem. 6, 273–293.Google Scholar
  65. Pitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulaev, O. I. (1990).FEBS. Lett. 262, 20–24.PubMedGoogle Scholar
  66. Postle, K., Nguyen, T. T., and Bertrand, K. P. (1984).Nucleic Acids Res. 12, 4848–4863.Google Scholar
  67. Punyiczki, M., Norman, J. A., Rosenberg, A. (1993).Biophys. Chem. 47, 9–19.Google Scholar
  68. Somogyi, B., and Lakos, Zs. (1993).J. Photochem. Photobiol. B: Biol. 18, 3–16.Google Scholar
  69. Somogyi, B., Norman, J. A., Punyiczki, M., and Rosenberg, A. (1992).Biochim. Biophys. Acta 1119, 81–89.PubMedGoogle Scholar
  70. Spencer, P. S., and Schaumburg, H. H. (1974).Can. J. Neurol. Sci. 33, 151–169.Google Scholar
  71. Tallmadge, D. H., Heubner, J. S., and Borkman, R. F. (1989).Photochem. Photobiol. 49, 381–386.PubMedGoogle Scholar
  72. Tovar, K., and Hillen, W. (1991).Meth. Enzymol. 208, 54–63.PubMedGoogle Scholar
  73. Wissman, A., Meier, I., Wray, L. V., Geissendorfer, M., and Hillen, W. (1986).Nucleic Acids Res. 14, 4253–4266.PubMedGoogle Scholar
  74. Wray, L. V., and Reznikoff, W. S. (1983).J. Bacteriol. 156, 1188–1191.PubMedGoogle Scholar
  75. Yang, H.-L., Zubay, G., and Levy, S. B. (1976).Proc. Natl. Acad. Sci. USA 73, 1509–1512.PubMedGoogle Scholar
  76. Yura, K., Tomoda, S., and Go, M. (1993).Protein Eng. 6, 621–628.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Jean-Alain Bousquet
    • 1
  • Norbert Ettner
    • 2
  1. 1.Laboratoire de Biophysique, Faculté de Pharmacie de StrasbourgUniversité Louis Pasteur, CNRS URA 491Illkirch-Graffenstaden
  2. 2.Lehrsthul für MikrobiologieInstitut für Mikrobiologie und Biochemie der Friedrich-Alexander UniversitätErlangenGermany

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