Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer.
The interaction of Tet repressor protein with the inducer tetracycline was studied by fluorescence measurements, equilibrium dialysis and nitrocellulose filter binding. The repressor-tetracycline complex was formed from two molecules of tetracycline and one Tet repressor dimer. Formation of the complex requires divalent cations, and results in drastic effects upon the fluorescence spectra of both compounds. The fluorescence of Tet repressor was quenched about 70%, while that of tetracycline was increased between three- and eightfold, depending upon pH. In addition, the emission maximum of the protein was shifted from 330 to 340 nm, and the excitation maximum of tetracycline dropped from 380 to 370 nm. The latter shift is accompanied by a similar change in the absorption spectra. An analogous effect was observed upon changing the environment of the drug by the addition of sodium dodecyl sulphate. These results suggest that tetracycline binds to a hydrophobic region of the protein. A new excitation band in the fluorescence spectrum of the complex is observed. This presumably arises from energy transfer from a tryptophan to the drug. The association rate constant for formation of the complex is 3.3(+/- 0.3) X 10(5) M-1 s-1, and the equilibrium association constant is 2.8(+/- 0.5) X 10(9) M-1. These results are discussed with respect to the biological function of the Tet repressor.
D002413:Cations, Divalent; D004734:Energy Metabolism; D004926:Escherichia coli / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013752:Tetracycline / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
D002413:Cations, Divalent; D012097:Repressor Proteins; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline