Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline.
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.
Hansen
D
D
Lehrstuhl für Mikrobiologie, Instut für Mikrobiologie und Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Federal Republic of Germany.
Altschmied
L
L
Hillen
W
W
D001426:Bacterial Proteins / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010649:Phenylalanine; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013752:Tetracycline / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014364:Tryptophan
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D012097:Repressor Proteins; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D010649:Phenylalanine; D014364:Tryptophan; D004247:DNA; D013752:Tetracycline