Fluorescence and phosphorescence study of Tet repressor-operator interaction.

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O1 and tet O2 operators. Binding of Tet R to tet O1 operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O2 operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O1 and that with the O2 operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 x 10(9) M(-1) sec(-1). In contrast to the Tet R-tet O2 complex, the Stern-Volmer acrylamide quenching constant Ksv of the complex with tet O1 operator changes from 7.5 M(-1) at 5 mM NaCl to 22 M(-1) at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0-0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0-0 vibronic bands of Trp 43 to 413 and 412 nm for tet O1 and tet O2 operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0-5.5 to 3.5-3.3 sec. The thermal phosphorescence quenching profile ranged from -200 degrees C to -20 degrees C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O1 and tet O2 operators.