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1 May 2012 Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of Irradiated E. coli Lactose Repressor
Stephane Goffinont, Sandrine Villette, Melanie Spotheim-Maurizot
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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.

Stephane Goffinont, Sandrine Villette, and Melanie Spotheim-Maurizot "Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of Irradiated E. coli Lactose Repressor," Radiation Research 177(6), 738-742, (1 May 2012).
Received: 6 December 2012; Accepted: 1 January 2012; Published: 1 May 2012

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