Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.Abbreviations CRP cyclic AMP receptor protein - NATA N-acetyltryptophanamide - FQRS fluorescence-quenching-resolved spectra - FDCD fluorescence-detected circular dichroism - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - FPLC fast protein liquid chromatography     

A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.     

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.     

Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.     

Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein

Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.     

Interdomain interaction of cyclic AMP receptor protein in the absence of cyclic AMP

Interdomain interaction of apo-cyclic AMP receptor protein (apo-CRP) was qualified using its isolated domains. The cAMP-binding domain was prepared by a limited proteolysis, while the DNA-binding domain was constructed as a recombinant protein. Three different regions making interdomain contacts in apo-CRP were identified by a sequence-specific comparison of the HSQC spectra. The results indicated that apo-CRP possesses characteristic modules of interdomain interaction that are properly organized to suppress activity and to sense and transfer the cAMP binding signals. Particularly, the inertness of the DNA-binding motif in apo-CRP was attributable to the participation of F-helices in the interdomain contacts.     

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.     

Structure and dynamics of the modular halves of Escherichia coli cyclic AMP receptor protein

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains. To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides. The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets. The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices. The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209. The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP. Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP. In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific. Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding. The acquisition of this unique property is intimately associated with the dynamics of the molecule.     

Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.