The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys 178 , localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP⅐cAMP 2 ) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: k c ‫؍‬ 3.62 s ؊1 and k ؊c ‫؍‬ 3.13 s ؊1 for CRP T127I and k c ‫؍‬ 0.42 s ؊1and k ؊c ‫؍‬ 0.78 s ؊1 for CRP S128A. These values can be compared with k c ‫؍‬ 9.7 s ؊1 and k ؊c ‫؍‬ 0.31 s ؊1 for wildtype CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP⅐cAMP 4 complex results from displacement of equilibrium between the two forms of the CRP⅐cAMP 2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser 128 and Thr 127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP.The cAMP receptor protein (CRP) 1 regulates the expression of Ͼ100 genes in Escherichia coli. CRP is a dimeric protein composed of two identical subunits of 209 amino acid residues with a molecular mass of 22.6 kDa (1, 2). Each subunit is folded into two distinct domains (3). The larger N-terminal domain contains a binding site for cAMP in the anti-conformation, and the smaller C-terminal domain contains a helix-turn-helix (HTH) motif responsible for DNA recognition and binding. The two domains are connected with a short hinge region composed of residues 135-138. Crystal structure studies have shown that the protein additionally possesses a second binding site (located between the hinge and the turn of HTH) that binds cAMP in the syn-co...

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The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or gal promoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9 x 10(6) M(-1) for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of gal promoter it made this interaction specific (apparent binding constant 2.93 x 10(7) M(-1)). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14 x 10(6) M(-1) for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP-lac promoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP-gal promoter interaction.