title: Allosteric control of cAMP receptor binding dynamics.
abstract: The intrinsic fluorescence of the cyclic AMP receptor is a sensitive indicator of the reaction with DNA, but signals are perturbed by a photoreaction. A ratio procedure is shown to be useful for correction. The reaction of the protein with DNA indicated by corrected transients extends over a broad time range not only at low salt concentrations but also at physiological salt concentrations. The initial binding step can be recorded preferentially at low salt pH 7 and is shown to be very similar for specific and nonspecific DNA. The rate constant for initial binding at 13.5 mM salt pH 7 is 2 × 10(8) M(-1) s(-1). Slow reaction steps up to times of several hundred seconds are observed both at low and high salt; the magnitude and sign of fluorescence amplitudes are strongly dependent on salt and pH. At 100 mM salt pH 8, the slow reaction step observed for the binding of the cyclic AMP receptor protein to promoter DNA is strongly shifted to longer times upon reduction of the cAMP concentration. The observed cAMP dependence is described quantitatively by a model implying that binding of the receptor to promoter DNA requires two cAMP molecules per protein dimer and is not consistent with a model assuming that a single cAMP is sufficient for activation. The rate constant for binding of the protein·dimer·(cAMP)(2) complex to the promoter is 1.3 × 10(8) M(-1) s(-1), close to the limit of diffusion control. Equilibration of specific complexes takes ~100 s at physiological concentrations of the reaction components.
authors.affiliation: Max Planck Institut für biophysikalische Chemie, 37077 Göttingen, Germany. dpoersc@gwdg.de
authors.forename: Dietmar
authors.initials: D
authors.lastname: Porschke
mesh_terms: D000494:Allosteric Regulation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000378:metabolism*; D012492:Salts / Q000737:chemistry
chemical_list: D011953:Receptors, Cyclic AMP; D012492:Salts; D004247:DNA