| Abstract
| - Fluorescence quenching was used to study thesite-specific binding of the Escherichia coliregulatory protein TyrR to a fluoresceinated oligonucleotide(9F30A/30B) containing a TyrR bindingsite. The equilibrium constant for the interaction(KL) was measured as a function of temperatureandsalt concentration in the presence and absence of ATPγS, a specificligand for TyrR. Fluorescence titrationsyielded a KL value of 1.20 × 107M-1 at 20 °C, which was independent of theacceptor (9F30A/30B)concentration in the range 5−500 nM, indicating that the systemexhibits true equilibrium binding. Clarkeand Glew analysis of the temperature dependence of binding revealed alinear dependence of R ln KLontemperature in the absence of ATPγS. The thermodynamicparameters obtained at 20 °C (θ) were= −35.73 kJ mol-1,= 57.41 kJ mol-1, and= 93.14 kJ mol-1. Saturating levelsofATPγS (200 μM) strengthened binding at all temperatures andresulted in a nonlinear dependence of Rln KL on temperature. Thethermodynamic parameters characterizing binding under these conditionswere= −39.32 kJ mol-1,=37.16 kJ mol-1,= 76.40 kJ mol-1, and= −1.03 kJ mol-1K-1. Several conclusions were drawn fromthese data. First, binding is entropically driven at 20 °Cinboth the presence and absence of ATPγS. This can partly beaccounted for by counterions released fromthe DNA upon TyrR binding; in the absence of ATPγS and divalentcations, the TyrR−9F30A/30Binteraction results in the release of two to three potassium ions.Second, the more favorablevalue,and hence tighter binding observed in the presence of ATPγS, isprimarily due to a decrease in(−20.3 kJ mol-1), which overcomes anunfavorable decrease in(−16.7 kJ mol-1). Third,thenegativevalue obtained in the presence of ATPγS indicates that the binding ofATPγS favors aconformational change in TyrR upon binding to 9F30A/30B, yielding amore stable complex.
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