Journal of Protein Chemistry

, Volume 14, Issue 8, pp 739–746

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

  • Zofia Blicharska
  • Zygmunt Wasylewski
Article

DOI: 10.1007/BF01886913

Cite this article as:
Blicharska, Z. & Wasylewski, Z. J Protein Chem (1995) 14: 739. doi:10.1007/BF01886913

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.

Key words

Trp repressorfluorescence quenchingresolved spectra

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

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Zofia Blicharska
    • 1
  • Zygmunt Wasylewski
    • 1
  1. 1.Department of Physical Biochemistry, Institute of Molecular BiologyJagellonian UniversityKrakowPoland