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Research has been conducted on Escherichia coli cyclic AMP receptor protein. The intermolecular communication mechanism and the covalent linkage properties have been investigated via the study of the functional and structural properties of the protein's DNA- and cyclic AMP-binding domains and the details are presented.

An iron-EDTA-mediated protein footprinting technique was utilized in the investigation of the conformational changes in the catalytic subunit of the cyclic AMP-dependent protein kinase upon substrate binding. Solvent-accessible enzyme surfaces as well as the structural dynamics of the catalytic domain were monitored during the footprinting process. Results reveal that the structural changes in the catalytic domain involve the transition from an open to a close conformation.

Research was conducted to determine the structural elements associated in the coupling of interfacial interactions by glycine 141 mutation in Escherichia coli cAMP receptor protein. Wild-type caMP receptor proteins were determined from temperature-induced Escherichia coli cells while their quaternary structure was monitored by sedimentation equilibrium. Results showed that the mutation differentially perturbed the network involving the interfacial relations between subunits and domains.

The communication between domains induced by cAMP binding can be dissociated from the proper subunit realignment of the cAMP receptor protein (CRP) dimer that is necessary for the activation of CRP. The mutations at position 128 were analyzed in order to identify the partial reactions in the activation pathway. The results indicate that mutations of Ser 128 to Ala or Pro does not disturb the overall secondary structure as monitored by circular synchronism in any significant ways.

Type-II DNA-(cytosine-5)-methyltransferase (M.HhaI) gene was transfected and overexpressed in Escherichia coli and purified using a salt back-extraction technique. The extracted M.HhaI can be isolated as pure molecules or complexed with S-adenosylmethionine, its cofactor. The cofactor-bound form can methylate nucleic acid. The enzyme kinetics of the purified M.HhaI were also reported. Crystallographic analysis of the enzyme revealed its growth along a monoclinic space group. Dimensions for each unit cell are presented.

Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.