Fluorescence and phosphorescence study of tet repressor-operator interaction

Fluorescence and phosphorescence measurements have been carried out on sing le-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 tw o 20-bp DNA sequences of tet O-1 and tet O-2 operators. Binding of Tet R to tet O-1 operator leads to a 78% decrease of the repressor fluorescence int ensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nn. Upon binding of Tet R to tet O-2 operator, the Trp 43 fl uorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acry lamide, performed at low ionic strength indicate that in both the complex o f Tet R with the O-1 and that with the O-2 operator, Trp 43 is moderately b uried, 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 O-2 complex, the Stern-Vol mer acrylamide quenching constant K-sv of the complex with tet O-1 operator changes from 7.5 M-1 at 5 mM NaCl to 22 M-1 at 200 mM NaCl, indicating dif ferent exposures of Trp 43 in the two complexes in solutions of higher ioni c 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 t he ret operators, we observed red shifts of 0-0 vibronic bands of Trp 43 to 413 and 412 nm for tet O-1 and tet O-2 operator, respectively, and the pho sphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0-5.5 t o 3.5-3.3 sec. The thermal phosphorescence quenching profile ranged from -2 00 degrees C to -20 degrees C, and differed drastically for the two complex es, suggesting different dynamics of the microenvironment of the Trp 43 res idue. The luminescence data for Trp 43 of Tet R suggest that the recognitio n helix of Tet R interacts in different fashions with the tet O-1 and tet O -2 operators.