Thermodynamics of the Interaction of the Escherichia coli Regulatory Protein TyrR with DNA Studied by Fluorescence Spectroscopy

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 × 107 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 = −35.73 kJ mol-1, = 57.41 kJ mol-1, and = 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 = −39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = −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 value, and hence tighter binding observed in the presence of ATPγS, is primarily due to a decrease in (−20.3 kJ mol-1), which overcomes an unfavorable decrease in (−16.7 kJ mol-1). Third, the negative 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.