Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (K(L)) was measured as a function of temperature and salt concentration in the presence and absence of ATPγS, a specific ligand for TyrR. Fluorescence titrations yielded a K(L) value of 1.20 x 10 7 M -1 at 20°C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln K(L) on temperature in the absence of ATPγS. The thermodynamic parameters obtained at 20°C (θ) were ΔG(θ)/°= -35.73 kJ mol -1, ΔH(θ)/°= 57.41 kJ mol -1, and TΔS(θ)/°= 93.14 kJ mol -1. Saturating levels of ATPγS (200 μM) strengthened binding at all temperatures and resulted in a nonlinear dependence of R ln K(L) on temperature. The thermodynamic parameters characterizing binding under these conditions were ΔG(θ)/°= -39.32 kJ mol -1, ΔH(θ)/°= 37.16 kJ mol -1, TΔS(θ)/°= 76.40 kJ mol -, and ΔC(pθ)/°= -1.03 kJ mol -1 K -1. Several conclusions were drawn from these data. First, binding is entropically driven at 20°C in both the presence and absence of ATPγS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPγS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable ΔG(θ)/°value, and hence tighter binding observed in the presence of ATPγS, is primarily due to a decrease in ΔH(θ)/°(-20.3 kJ mol -1), which overcomes an unfavorable decrease in TΔS(θ)/°(-16.7 kJ mol -1). Third, the negative ΔC(pθ)/°value obtained in the presence of ATPγS indicates that the binding of ATPγS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.