PMC

(A) Experimental scattering data for TraA (○) and TraAN₂₄₆ (△) in Tris/NaCl buffer (see the Experimental section). (B) PDDFs of TraA (○) and TraAN₂₄₆ (△) solutions derived from the scattering functions; the theoretical PDDFs are shown for a sphere corresponding to the TraA monomer and the TraA dimer. (C) Size distribution function of TraAN₂₄₆ solution, weighted by particle volume.

Increasing amounts of TraA (A and C) or TraAN₂₄₆ (B and D) were incubated with 0.5 nM DNA at 42 °C for 30 min and then loaded on to a 10% (w/v) native polyacrylamide gel. The EMSAs were performed with the 42-mer oligonucleotide (A and B) and the hairpin_nic oligonucleotide (C and D).

Samples of 0.5 mg/ml TraA (A) or TraAN₂₄₆ (B) were incubated with increasing glutaraldehyde concentrations, then the products were loaded on to SDS/18%-polyacrylamide gels, electrophoresed at a constant voltage of 180 V and stained with Coomassie Brilliant Blue. Lane 1, no glutaraldehyde; lane 2, 0.001% glutaraldehyde; lane 3, 0.01%; lane 4, 0.05%; lane 5, 0.1%; lane 6, SeeBlue® plus2 prestained protein standards (Invitrogen).

Samples were applied to an SDS/12% (w/v) (TraA; A) or 18% (w/v) (TraAN₂₄₆; B) polyacrylamide gel stained with Coomassie Brilliant Blue. Lanes 1 and 5, Mark12™ protein standard (Invitrogen, Carlsbad, CA, U.S.A.); lane 2, clarified lysate; lane 3, eluate after the HiTrap Chelating Ni²⁺-charged column (∼50 μg of TraA; ∼4 μg of TraAN₂₄₆); lane 4, eluate after the HiTrap heparin column (∼10 μg of TraA; ∼3 μg of TraAN₂₄₆).

A 0.7% (w/v) agarose gel was run in TAE buffer (40 mM Tris/acetate, 1 mM EDTA, pH 8.0), and stained with ethidium bromide. Lane 1, pVA2241 without protein; lane 2, pVA2241 with 20-fold molar excess TraA; lane 3, pVA2241 with 80-fold excess TraA; lane 4, pVA2241 with 20-fold excess TraAN₂₄₆; lane 5, pVA2241 with 80-fold excess TraAN₂₄₆; lane 6, MassRuler™ DNA Ladder [high range: 10, 8, 6, 5, 4 and 3 kb (MBI Fermentas, St. Leon-Rot, Germany)]; lane 7, RSF1010 with 80-fold excess TraA; lane 8, RSF1010 with 80-fold excess TraAN₂₄₆; lane 9, RSF1010 without protein. oc, open circular plasmid DNA; ccc, covalently closed circular plasmid DNA.

The DNA fragment of the original oriT_pIP501 sequence is shown on a grey background. Position numbers refer to GenBank accession no. L39769. The experimentally determined nick site [] is marked with a vertical arrow. Horizontal arrows indicate an inverted repeat, potentially generating a secondary structure. The exchanged base for generating a perfect hairpin is shown in bold. K_D values are means±S.D. of three independent measurements (except for the hairpin oligonucleotide with TraAN₂₄₆ – two measurements). K_D values were determined with the liquid scintillation counter (see Experimental section). *The K_D value for the TraAN₂₄₆/hairpin A→C complex was calculated using AlphaEase software. n.d., not determined.

Far-UV CD spectra of the proteins were recorded in a 0.02 cm cylindrical cell with a scan rate of 50 nm/min at 20 °C. The spectra represent the accumulation of three repetitive scans and are normalized for mean residue weight and concentration of the respective protein. (A) TraA, TraAN₂₄₆ and complex (TraAN₂₄₆+42-mer) spectra recorded at 20 °C. (B) Heat-induced unfolding of TraA, TraAN₂₄₆ and the complex: [θ]₂₀₈ values recorded during the scan were normalized for the room temperature values ([θ]₂₀₈²⁰) in order to yield comparable unfolding curves. (C, D) Heat denaturation of TraA (C) and TraAN₂₄₆ (D). Spectra of the samples were taken at 20 °C, and following heating to 60 °C, and then cooling to 20 °C.