The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor. The affinity of CRP for a specific DNA sequence is significantly enhanced as a consequence of the binding of the allosteric effector, cAMP. The hinge region, particularly residues 136 and 138, connecting the cAMP and DNA binding domains of CRP has been proposed to play essential roles in transmitting the allosteric signals. To probe the specific role of residue 138, eight D138 mutants and wild-type CRP were tested for their ability to bind the lac26 and gallac26 promoter sequences in this study. A correlation was established between DNA binding affinity and side chain solvation free energy, namely, an increasing specific DNA affinity with an increasing hydrophilicity of the side chain of residue 138. In addition, a linear correlation was found between DNA binding affinity and the energetics of subunit assembly. The ability of CRP to distinguish between cAMP and cGMP as an allosteric activator of DNA binding is weakened with higher energetics of subunit assembly. This correlation indicates that quaternary constraint leads to a constraint of the DNA binding domain. This observation is consistent with the concept that an optimum quaternary structural constraint is important in CRP exhibiting its allosteric properties. The stability of CRP was monitored by Trp fluorescence and circular dichroism in the presence of guanidine hydrochloride. These spectroscopic data revealed nonidentical denaturation profiles. Since the Trp residues are located exclusively in the β-roll cyclic nucleotide binding domain, the denaturation profiles reveal the stability of the β-roll structure. This study produces another linear correlation between DNA binding affinity in the presence of cAMP and cGMP and the stability of the β-roll; namely, the stability of the β-roll structure leads to a decrease in DNA binding affinity. All these correlations indicate the importance of structural stability and dynamics in the ability of CRP to manifest its intrinsic allosteric properties.
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