Abstract
Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- TrpR:
-
tryptophan aporepressor fromE. coli
- TrpR W19F:
-
TrpR mutant with phenylalanine substituted for tryptophan at position 19
- TrpR W99F:
-
TrpR mutant with phenylalanine substituted for tryptophan at position 99
- FQRS:
-
fluorescence-quenching-resolved spectra
- FPLC:
-
fast protein liquid chromatography
References
Alcala, J. R., Gratton, E., and Prendergast, F. G. (1987).Biophys. J. 51, 925–936.
Beecham, J. C., and Brand, L. (1985).Annu. Rev. Biochem. 54, 43–71.
Eftink, M. R., Ramsay, G. D., Burns, L., Maki, A. H., Mann, C. J., Matthews, C. R., and Ghiron, C. A. (1993).Biochemistry 32, 9189–9198.
Gunsalus, R. P., and Yanofsky, C. (1980).Proc. Natl. Acad. Sci. USA 77, 7117–7121.
Liu, Y. C., and Matthews, S. K. (1993).Biochemistry 32, 10532–10542.
Luisi, B. F., and Sigler, P. B. (1990).Biochim. Biophys. Acta 1048, 113–126.
Royer, C. A. (1992).Biophys. J. 63, 741–750.
Stryjewski, W., and Wasylewski, Z. (1986).Eur. J. Biochem. 158, 547–553.
Wasylewski, Z., Koloczek, H., and Wasniowska, A. (1988).Eur. J. Biochem. 172, 719–724.
Zhang, R., Joachimiak, A., Lawson, C. L., Schevitz, R. W., Otwinowski, Z., and Sigler, P. B. (1987).Nature 327, 591–597.
Xhao, D., Arrowsmith, C. H., Jia, X., and Jardetzky, O. (1993).J. Mol Biol. 229, 735–746.
Zurawski, G., Gunsalus, R. P., Brown, K. D., and Yanofsky, C. (1981).J. Mol. Biol. 145, 47–53.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Blicharska, Z., Wasylewski, Z. Fluorescence quenching studies of Trp repressor using single-tryptophan mutants. J Protein Chem 14, 739–746 (1995). https://doi.org/10.1007/BF01886913
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01886913