Kinetic studies of cAMP-induced propagation of the allosteric signal in the cAMP receptor protein from Escherichia coli with the use of site-directed mutagenesis.

The cyclic AMP receptor protein (CRP) - general transcription factor in Escherichia coli - changes their conformation after the cAMP binding. For CRP mutants bearing the amino acids substitutions in position 138 located within the hinge region, the fluorescence stopped-flow measurements have been employed to study the kinetics of the conformational changes. By using two naturally appearing Tryptophan residues (W13, W85) localized nearby the ligand binding pocket and 1,5-I-AEDANS-labeled C178 localized in the helix-turn-helix (HTH) motif within the C-terminal domain as a fluorescence probes, we observed a first and a consensus steps of CRP-cAMP association, respectively. The collected data suggest that the kinetic parameters determined for mutants, reflect a component of the conformational change occurring in the native protein. Therefore, the independent association of two cAMP molecules to the wt protein is followed by at least a three-step conformational change which alters the surroundings of HTH motifs.