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 (α-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by β-sheets. The C-terminal DNA-binding domain, designated as β-CRP, consists of mostly α-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 β-CRP are from 108 to 209. The secondary structures of α-CRP and β-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 β-CRP is more dynamic than α-CRP. In an earlier study, it was shown that α-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. β-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.
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