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 olig onucleotide (9F30A/30B) containing a TyrR binding site. The equilibriu m constant for the interaction (K-L) was measured as a function of tem perature and salt concentration in the presence and absence of ATP gam ma S, a specific ligand for TyrR. Fluorescence titrations yielded a K- L value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicati ng that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear de pendence of R ln K-L on temperature in the absence of ATP gamma S. The thermodynamic parameters obtained at 20 degrees C (theta) were Delta G(theta)degrees = -35.73 kJ mol(-1), Delta H(theta)degrees = 57.41 kJ mol(-1), and T Delta S(theta)degrees 93.14 kJ mol(-1). Saturating leve ls or ATP gamma S (200 mu M) 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 condition s were Delta G(theta)degrees = -39.32 kJ mol(-1), Delta H(theta)degree s = 37.16 kJ mol(-1), T Delta S(theta)degrees = 76.40 kJ mol(-1), and Delta C(p theta)degrees = -1.03 kJ mol(-1) K-1. Several conclusions we re drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATP gamma S. This can pa rtly be accounted for by counterions released from the DNA upon TyrR b inding; in the absence of ATP gamma S and divalent cations, the TyrR-9 F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable Delta G(theta)degrees value, and henc e tighter binding observed in the presence of ATP gamma S, is primaril y due to a decrease in Delta H(theta)degrees (-20.3 kJ mol(-1)), which overcomes an unfavorable decrease in T Delta S(theta)degrees (-16.7 k J mol(-1)). Third, the negative Delta C(p theta)degrees value obtained in the presence of ATP gamma S indicates that the binding of ATP gamm a S favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.