title: Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.
abstract: The function of the  E. coli  lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadron, 45071 Orléans, France.
authors.forename: Stephane
authors.initials: S
authors.lastname: Goffinont
authors.forename: Sandrine
authors.initials: S
authors.lastname: Villette
authors.forename: Melanie
authors.initials: M
authors.lastname: Spotheim-Maurizot
mesh_terms: D000409:Alanine / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli; D056968:Lac Repressors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics* / Q000528:radiation effects*; D009154:Mutation / Q000528:radiation effects; D055550:Protein Stability / Q000528:radiation effects; D017433:Protein Structure, Secondary / Q000528:radiation effects; D017434:Protein Structure, Tertiary / Q000528:radiation effects; D013696:Temperature
chemical_list: D056968:Lac Repressors; D004247:DNA; D000409:Alanine