Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH.
The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy. The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size. The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP. This number is larger than that previously found in the specific binding process. The effect of pH on the non-specific binding was investigated. The number of ion pairs does not vary between pH 6 and 8. From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP. These are most probably the histidines 199 of each subunit. In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA.
Giraud-Panis
M J
MJ
Centre de Biophysique Moléculaire, Orléans, France.
Toulmé
F
F
Blazy
B
B
Maurizot
J C
JC
Culard
F
F
D000242:Cyclic AMP; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D002373:Cyclic AMP Receptor Protein; D006639:Histidine; D004247:DNA; D000242:Cyclic AMP