Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native⇄2(CRP) Native⇄2(CRP)Denatured. The ΔG values for dissociation (ΔGd) and unfolding (ΔGu) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 ± 0.6 and 7.2 ± 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar ΔGd values of 10.1 ± 0.4 and 9.5 ± 0.4 kcal/mol, respectively. Results from this study in conjunction with cristallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization. ΔGu for both S-CRP and CH-CRP is about 8.7 ± 0.4 kcal/mol, which is about 1.5 kcal/mol more positive than that of CRP. This implies that the isolated cAMP binding domain is more stable than the whole CRP subunit which contains both the DNA and the cAMP binding domain. These results can only be observed if there is interaction between these domains.
|Original language||English (US)|
|Number of pages||10|
|State||Published - 1993|
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