Abstract
Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.
Highlights
From the Lehrstuhlfur Mikrobiologie, Znstitut fur Mikrobiologie und Biochemieder Friedrich-Alexander-UniuersitdErlangenNiirnberg, Staudtstrasse 5, 8520 Erlangen, Federal Republic of Germany
The fluorescence emission spectra ofboth tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched tozero and Trp-43 to only 50%.It is concluded thatTrp-75 is in the vicinity of the inducerbindingsite
The different fluorescence emission spectra of bothtryptophan residues depend on the native structure of Tet repressor
Summary
General Methods-All chemicals were of the highest purity available purchased from either Merck (Darmstadt), Serva (Heidelberg), Fluka (Buchs), Sigma, or Roth (Heidelberg).Tetracycline hydrochloride was from Fluka (Buchs) and was used without further purification. The fluorescence emission spectra of denatured Tet repressors were recorded at a concentration of 0.5 p~ in 6 M guanidinium hydrochloride, pH 5.5.All other conditions were as described above. The Faand F values were measured as the difference of the respective fluorescence emission intensities minus the emission of the F43F75 double mutant determined at identical concentrations. This procedure is based on the assumption that fluorescence of the F43F75 protein results mainly from the background of contaminatingproteins.
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