Photochemical modifications of lac repressor--II. Tryptophan photochemistry as a probe in studying the allosteric behaviour of the protein.
Irradiation of lac repressor under aerobic conditions in the near UV region (295-400 nm) decreases the Trp fluorescence of the protein. A total loss of fluorescence corresponds to the destruction of all tryptophanyl residues. Irradiation with light of wavelength between 250 and 400 nm quenches fluorescence completely when only half of the Trp residues ae destroyed. An internal photodynamic effect, in which N-formylkynurenine, a principal photoproduct of Trp, sensitizes further the destruction of the other Trp residues, accounts for our results. Experiments performed in the presence of sodium azide suggest that singlet oxygen is not involved in the destruction of Trp, but may be responsible for histidine degradation. Irradiating the repressor complexed with non-operator E. coli DNA has the same effect on Trp residues as irradiating repressor alone. On the contrary, when repressor is complexed to lac operator, both tryptophanyl residues seem to be destroyed simultaneously. This indicates that binding of specific operator DNA at the DNA site induces changes in the environment of the tryptophanyl residues (mainly tor Trp 220) which cannot further transfer in excitation energy to the photoproduct of the other Trp. A prolonged irradiation destroys the complex, leading to the same result observed for non-specific complex or for repressor alone. These results are discussed in terms of the proximity of Trp from the inducer binding site and the allosteric behaviour of the repressor.
Spodheim-Maurizot
M
M
Culard
F
F
Charlier
M
M
D000494:Allosteric Regulation; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D012097:Repressor Proteins / Q000378:metabolism / Q000528:radiation effects*; D014157:Transcription Factors / Q000528:radiation effects*; D014364:Tryptophan / Q000528:radiation effects*
D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan