Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding

We analysed the conformational states of free, tet operator-bound and anhyd rotetracycline-bound Tet repressor employing a Trp-scanning approach. The t wo wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, repre senting part of an unstructured loop and the N-terminal six residues of alp ha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycl ine-binding constants were decreased up to 7.5-fold when Trp was in positio ns 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching r ate constants with acrylamide and anisotropies in the free and tet operator -complexed proteins. Trp fluorescence emission decreased drastically upon a te binding, mainly due to energy transfer. For all proteins, either free, t et operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surf aces of the respective Trp side chains were calculated and compared with th e quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in a-helix 9 become more buried upon the induction of Tet R by anhydrotetracycline.