Kinetic studies of cAMP-induced allosteric changes in cyclic AMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric Ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics o f the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-1 78 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP(2) complex, i.e, after saturatio n of the high affinity sites. The observed conformational change occurs acc ording to sequential model of allostery and is described by rate constants: k(c) = 9.7 +/- 0.1 s(-1) and k(-c) = 0.31 +/- 0.05 s(-1), for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP(4) complex and results from displacement of equilibr ium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affi nity sites of one of these forms only. The observed conformational change o ccurs according to concerted model of allostery and is described by rate co nstants: k(on) = 28 +/- 1.5 s(-1) and k(off) = 75.5 +/- 3 s(-1). Results of experiments using single-tryptophan-containing CRP mutants indicate that T rp-85 is mainly responsible for the observed total change in intrinsic fluo rescence of wild-type CRP.