title: Fluorescence quenching studies of Trp repressor-operator interaction.
abstract: Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern-Volmer quenching constant equal to about 2.0-2.3 M(-1). The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern-Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern-Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M(-1). The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.
authors.affiliation: Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
authors.forename: Z
authors.initials: Z
authors.lastname: Blicharska
authors.forename: Z
authors.initials: Z
authors.lastname: Wasylewski
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli; D005453:Fluorescence; D010649:Phenylalanine; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D010649:Phenylalanine; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins