PROXIMITY MAPPING OF THE TET REPRESSOR TETRACYCLINE FE2-PEROXIDE MEDIATED PROTEIN CLEAVAGE( COMPLEX BY HYDROGEN)

We demonstrate in a quantitative in vitro induction assay that tetracy cline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor -tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe2+- dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in t he presence of the drug, whereas about 1% cleavage is obtained after 3 0 min in the presence of Fe2+ without tetracycline. Cleavage is not in hibited by several radical scavengers, suggesting a highly localized r eactivity of the redox-active oxo intermediates in the proximity of th e Fe2+-tc chelater where they are generated, The products can be separ ated by HPLC only after denaturation, indicating that the complex is n ot disrupted by cleavage. Residues at which the cleavage takes place a re identified using the masses of the fragments determined by electros pray mass spectrometry and their N-terminal sequences. The major cleav age site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cle aved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycli ne-Mg2+ complex. We discuss potential reaction mechanisms leading to p rotein cleavage.