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 of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 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 saturation of the high affinity sites. The observed conformational change occurs according 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 equilibrium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants: 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 Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP.
Małecki
J
J
Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Al. Mickiewicza 3, 31-120 Kraków, Poland.
Polit
A
A
Wasylewski
Z
Z
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000187:drug effects*; D000242:Cyclic AMP / Q000494:pharmacology*; D002373:Cyclic AMP Receptor Protein / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000187:drug effects*; D004926:Escherichia coli; D017022:Flow Injection Analysis; D007700:Kinetics; D008962:Models, Theoretical; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D014364:Tryptophan; D000242:Cyclic AMP