Abstract
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.
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Abbreviations
- Tris:
-
tris(hydroxymethyl)aminomethane
- DTT:
-
dithiothreitol
- FVSTetR:
-
engineered tetracycline repressor in which the Trp residue at the position 75 in the wild-type repressor TetR is replaced by a Phe residue
- H-t-H:
-
helixα-turn-helixα super-secondary structure
References
Altschmied, L., Baumeister, R., Pleiderer, K., and Hillen, W. (1988).EMBO J. 7, 4011–4017.
Baumeister, R., Muller, G., Hecht, B., and Hillen, W. (1992).Proteins 14, 168–177.
Beck, C. G., Mutzel, R., Barbe, J., and Muller, W. (1982).J. Bacteriol. 150, 633–642.
Berens, C., Altshmied, L., and Hillen, W. (1992).J. Biol. Chem. 267, 1945–1952.
Bertrand, K. P., Postle, K., Wray, L. V., and Reznikoff, W. S. (1983).Gene 23, 149–156.
Blatt, E., Husain, A., and Sawyer, W. H. (1986).Biochim. Biophys. Acta 871, 6–13.
Boczko, E. M., and Brooks, C. L. (1995).Science 269, 393–396.
Brennan, R. G., and Matthews, B. W. (1989).J. Biol. Chem. 264, 1903–1906.
Calhoun, D. B., Vanderkooi, J. M., and Englander, S. W. (1983).Biochemistry 22, 1533–1539.
Carell, R. W., Evans, O., and Stein, P. E. (1991).Nature 353, 576–578.
Carell, R. W., Evans, O., and Stein, P. E. (1993).Nature 364, 737.
Cavins, J. F., and Friedman, M. (1968).J. Biol. Chem. 243, 3357.
Chabbert, M., Hillen, W., Hansen, D., Takahashi, M., and Bousquet, J. A. (1992).Biochemistry 31, 1951–1960.
Danilveiante, M., Adomeniene, O., and Dienys, G. (1981).Org. Reactiv. 18, 217–224.
Dodd, I. B., and Eagan, J. B. (1987).J. Mol. Biol. 194, 557–564.
Eftink, M. R., and Ghiron, C. A. (1975).Proc. Natl. Acad. Sci. USA 72, 3290–3294.
Eftink, M. R., and Ghiron, C. A. (1976).Biochemistry 15, 672–680.
Eftink, M. R., and Ghiron, C. A. (1977).Biochemistry 16, 5546–5551.
Eftink, M. R., and Ghiron, C. A. (1981).Anal. Biochem. 114, 199–227.
Eftink, M. R., and Ghiron, C. A. (1984).Biochemistry 23, 6891–6899.
Eftink, M. R., and Ghiron, C. A. (1987).Biochem. Biophys. Acta 916, 343–349.
Eftink, M. R., and Selvidge, L. A. (1982).Biochemistry 21, 117–125.
Eftink, M. R., Wasylewsky, Z., and Ghiron, C. A. (1987).Biochemistry 26, 8338–8346.
Englander, S. W. (1975).Ann. N. Y. Acad. Sci. 244, 10–27.
Englander, S. W., and Kallenbach, N. R. (1983).Q. Rev. Biophys. 16, 521–655.
Follenius, A., and Gerard, D. (1983).Photochem. Photobiol. 38, 373–376.
Geisthardt, D., and Kruppa, J. (1987).Anal. Biochem. 160, 184–191.
Gratton, E., Jameson, D. M., Weber, G., and Alpert, B. (1984).Biophys. J. 45, 789–794.
Hagaman, K. A., and Eftink, M. R. (1984).Biophys. Chem. 20, 201–207.
Hansen, D., and Hillen, W. (1987).J. Biol. Chem. 262, 12269–12274.
Hansen, D., Altshmied, L., and Hillen, W. (1987).J. Biol. Chem. 262, 14030–14035.
Harrison, S. C., and Aggarval, A. K. (1990).Annu. Rev. Biochem. 59, 933–969.
Haynie, D. T., and Freire, E. (1993).Proteins Struct. Funct. Genet. 16, 115–140.
Hillen, W., and Berens, C. (1994).Annu. Rev. Microbiol. 48, 345–369.
Hillen, W., and Schollmeier, K. (1983).Nucleic Acids Res. 11, 525–539.
Hillen, W., and Unger, B. (1982).Nature 297, 700–702.
Hillen, W., Klock, G., Kaffenberger, I., Wray, L. V., and Reznikoff, W. S. (1982).J. Biol. Chem. 257, 6605–6613.
Hillen, W., Gatz, C., Altshmied, L., Schollmeier, K., and Meier, I. (1983).J. Mol. Biol. 169, 707–721.
Hillen, W., Schollmeier, K., and Gatz, C. (1984).J. Mol. Biol. 172, 185–201.
Hinrichs, W., Kisker, C., Düvel, M., Müller, A., Tovar, K., Hillen, W., and Saenger, W. (1994).Science 264, 418–420.
Ilich, P., and Prendergast, F. G. (1991).Photochem. Photobiol. 53, 445–453.
Isackson, P. J., and Bertrand, K. P. (1985).Proc. Natl. Acad. Sci. USA 82, 6226–6230.
Jameson, D. M., Gratton, E., Weber, G., and Alpert, B. (1984).Biophys. J. 45, 795–803.
Jordan, S. R., and Pabo, C. O. (1988).Science 242, 893–899.
Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., and Saenger, W. (1995).J. Mol. Biol. 247, 260–280.
Kleinschimdt, C., Tovar, K., Hillen, W., and Porschke, D. (1988).Biochemistry 27, 1094–1104.
Kleinschimdt, C., Tovar, K., and Hillen, W. (1991).Nucleic Acids Res. 19, 1021–1028.
Kulus, J. (1981).Biotechnol. Bioeng. 23, 2875.
Kurban, G. P., Gitlin, G., Bayer, E. A., Walchek, M., and Horowitz, P. M. (1990).J. Protein Chem. 9, 673–682.
Kuwajima, K. (1989).Proteins Struct. Funct. Genet. 6, 87–103.
Lakowicz, J. R. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York, pp. 111–153.
Lakowicz, J. R., and Weber, G. (1973).Biochemistry 12, 4161–4171.
Lakowicz, J. R., and Weber, G. (1973).Biochemistry 53, 445–453.
Lederer, H., Tovar, K., Baer, G., May, R. P., Hillen, W., and Heumann, H. (1989).EMBO J. 8, 1257–1263.
Matko, J., Tron, L., Balazs, M., Hevessy, J., Somogyi, B., and Damjanovich, S. (1980).Biochemistry 19, 5782–5786.
McGhee, J. D., and von Hippel, P. H. (1974).J. Mol. Biol. 86, 469–489.
Meier, I., Wray, L. W., and Hillen, W. (1988).EMBO J. 7, 567–572.
Merill, A. R., Palmer, L. R., and Szabo, A. G. (1993).Biochemistry 32, 6974–6981.
Mondragon, A., and Harrison, S. C. (1991).J. Mol. Biol. 219, 321–334.
Narasimhulu, S. (1991).Biochemistry 30, 9319–9327.
Ngo, T. T. (1976).Int. J. Biochem. 7, 193–197.
Niederweis, M., and Hillen, W. (1993).Electrophoresis 14, 693–698.
Nowak, M. W., and Berman, H. A. (1991).Biochemistry 30, 7642–7651.
Pitsyn, O. B. (1987).J. Protein Chem. 6, 273–293.
Pitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulaev, O. I. (1990).FEBS. Lett. 262, 20–24.
Postle, K., Nguyen, T. T., and Bertrand, K. P. (1984).Nucleic Acids Res. 12, 4848–4863.
Punyiczki, M., Norman, J. A., Rosenberg, A. (1993).Biophys. Chem. 47, 9–19.
Somogyi, B., and Lakos, Zs. (1993).J. Photochem. Photobiol. B: Biol. 18, 3–16.
Somogyi, B., Norman, J. A., Punyiczki, M., and Rosenberg, A. (1992).Biochim. Biophys. Acta 1119, 81–89.
Spencer, P. S., and Schaumburg, H. H. (1974).Can. J. Neurol. Sci. 33, 151–169.
Tallmadge, D. H., Heubner, J. S., and Borkman, R. F. (1989).Photochem. Photobiol. 49, 381–386.
Tovar, K., and Hillen, W. (1991).Meth. Enzymol. 208, 54–63.
Wissman, A., Meier, I., Wray, L. V., Geissendorfer, M., and Hillen, W. (1986).Nucleic Acids Res. 14, 4253–4266.
Wray, L. V., and Reznikoff, W. S. (1983).J. Bacteriol. 156, 1188–1191.
Yang, H.-L., Zubay, G., and Levy, S. B. (1976).Proc. Natl. Acad. Sci. USA 73, 1509–1512.
Yura, K., Tomoda, S., and Go, M. (1993).Protein Eng. 6, 621–628.
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Bousquet, JA., Ettner, N. A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study. J Protein Chem 15, 205–218 (1996). https://doi.org/10.1007/BF01887401
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DOI: https://doi.org/10.1007/BF01887401