Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.
The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site.
Mazier
S
S
Centre de Biophysique Moléculaire, CNRS, Orléans, France.
Villette
S
S
Goffinont
S
S
Renouard
S
S
Maurizot
J C
JC
Genest
D
D
Spotheim-Maurizot
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000528:radiation effects*; D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000528:radiation effects; D004295:Dihydroxyphenylalanine / Q000737:chemistry; D017510:Protein Folding; D011829:Radiation Dosage; D012097:Repressor Proteins / Q000737:chemistry / Q000528:radiation effects*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D004295:Dihydroxyphenylalanine