Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes

The complexes designed in this work combine the sequence-specific binding p roperties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-bindin g domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific bind ing in terms of the polyelectrolyte and hydrophobic effects. The roles of t he electrostatic interactions in binding to the cognate and noncognate sequ ences indicate that nonspecific binding is more sensitive to changes in sal t concentration, whereas the change in the heat capacity shows a greater se nsitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate b ind noncooperatively to the DNA. The characteristic thermodynamic parameter s of specific and nonspecific DNA binding by each of the DNA-binding domain s and their respective conjugates are presented.