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.
Górecki
Andrzej
A
Department of Physical Biochemistry, Jagiellonian University, Kraków, Poland. andrzej.gorecki@uj.edu.pl
Kepys
Barbara
B
Bonarek
Piotr
P
Wasylewski
Zygmunt
Z
D000494:Allosteric Regulation / Q000187:drug effects; D019943:Amino Acid Substitution / Q000187:drug effects; D000242:Cyclic AMP / Q000737:chemistry / Q000494:pharmacology*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009282:Naphthalenesulfonates; D015398:Signal Transduction / Q000187:drug effects*; D013816:Thermodynamics
D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D009282:Naphthalenesulfonates; C481662:crp protein, E coli; C006421:1,5-I-AEDANS; D000242:Cyclic AMP