Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.
Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.
Gillard
Nathalie
N
Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France.
Goffinont
Stephane
S
Buré
Corinne
C
Davidkova
Marie
M
Maurizot
Jean-Claude
JC
Cadene
Martine
M
Spotheim-Maurizot
Melanie
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000528:radiation effects*; D002588:Cesium Radioisotopes; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000528:radiation effects; D017665:Hydroxyl Radical / Q000528:radiation effects; D056968:Lac Repressors; D008715:Methionine / Q000528:radiation effects; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D020673:Protein Renaturation; D017433:Protein Structure, Secondary / Q000528:radiation effects; D012097:Repressor Proteins / Q000528:radiation effects*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000528:radiation effects
D001426:Bacterial Proteins; D002588:Cesium Radioisotopes; D004268:DNA-Binding Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D017665:Hydroxyl Radical; D014443:Tyrosine; D008715:Methionine