Installation and testing of a focusing mirror at beamline X28C for high flux x-ray radiolysis of biological macromolecules.

The NSLS X28C white-light beamline has been upgraded with a focusing mirror in order to provide increased x-ray density and a wide selection of beam shapes at the sample position. The cylindrical single crystal silicon mirror uses an Indalloy 51 liquid support bath as both a mechanism for heat transfer and a buoyant support to counter the effects of gravity and correct for minor parabolic slope errors. Calorimetric measurements were performed to verify that the calculated more than 200-fold increase in flux density is delivered by the mirror at the smallest beam spot. The properties of the focused beam relevant to radiolytic footprinting, namely, the physical dimensions of the beam, the effective hydroxyl radical dose delivered to the sample, and sample heating upon irradiation, have been studied at several mirror angles.

Operator recognition by the phage 434 cI repressor: MD simulations of free and bound 50-bp DNA reveal important differences between the OR1 and OR2 sites.

Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer. Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites. Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites. In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2). Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove. This structure is maintained because structural fluctuations are weak. Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1. Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions. The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites. Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2. Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites. Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein.