Figure 6.

FRET efficiency versus CopR concentration. With increasing CopR concentration the FRET efficiency of the doubly labeled 19 bp DNA fragment containing the minimum operator sequence decreases by ∼3%. The insert shows the quenching of the donor fluorescein by increasing protein concentration. The donor intensity in a donor-only sample decreases by ∼30% while the quenching in the fluorescein and rhodamine labeled sample is ∼2–3% less corresponding with the decreasing FRET efficiency in the complex. While the donor fluorescence quantum yield has a direct influence on the Forster distance R₀ (see Materials and Methods) and therefore an influence on the measured FRET efficiencies, this quenching effect has to be taken into account in calculating the change of the dye-to-dye distance in the free and bound DNA. Thus, the distance between the dyes attached to the DNA helix ends decreases from 6.4 ± 0.1 nm (R₀ = 5.0 nm) in the free DNA to 6.2 ± 0.1 nm (R₀ = 4.7 nm) in the complex with CopR indicating a slight bend of the DNA in the complex. The dissociation constant of the CopR–DNA complex in solution determined from the FRET efficiency and the quenching data was 1.6 ± 0.9 nM.