Conformational changes induced in the Tet repressor protein TetR(B) upon operator or anhydrotetracycline binding as revealed by time-resolved fluorescence spectroscopy on single tryptophan mutants

We have analyzed the tryptophan (trp) fluorescence-decay kinetics of single trp mutants of the Tet repressor protein in the free, the tet operator and anhydrotetracycline (atc)-bound states. The position of the single trp var ies between residues 164 and 171, in close proximity to one entrance of the tetracycline-binding pocket. A good fit of the trp fluorescence decay need ed generally three exponentials. The decay times vary with detection wavele ngth, the extent of this variation being correlated to the variation of the emission maximum. Quenching experiments with neutral (acrylamide), cationi c (N-methylpyridinium chloride) and anionic quencher (KI) support the inter pretation of the three fluorescence components within a conformer model. Op erator and ate binding change the ratio of the relative amplitudes of the m edium- and long-lived component, thus pointing to structural changes as ind icated also by the changes in decay time. Since the fluorescence decay is d ifferent between the free, atc- and operator-bound states we conclude that the protein structure is different in each of these three states. The fluor escence quenching constants reflect not only the variation in solvent expos ure with position, but also the fact that the net surface charge in this re gion is negative, because the quenching constants by the cationic quencher are up to 10-fold higher. The ate fluorescence appears to decay monoexponen tially with about the same decay time for all mutants, except W170, in whic h the trp residue sterically interferes with atc.