The normal functioning mode of E. coli cyclic AMP receptor (CRP) requires it to distinguish among the more than 20 specific DNA sequences and the various cyclic nucleotides present in the cellular milieu. Upon activation by cAMP, CRP binds to a specific DNA-site. Thus, the mode of CRP activity is a fine balance between diversity and specificity. However, what structural elements define diversity by modulating the interactions in the CRP-DNA interface? This laboratory has identified some mutant CRPs whose rank order of their affinity for a series of DNA sequences is not only a function of the site of mutation but also the specific identity of the bound cyclic nucleotide. Thus, CRP is prime for an in-depth study to elucidate the mechanism of modulation with the acquisition of both energetic and structural information. The loss of specificity for cAMP as an allosteric activator in these CRP mutants may be related to the additional cAMP binding site in the DNA binding domain identified recently by crystallography. Thus, new direct methods are being developed to yield ligand binding isotherms and, in particular, site-specific ones to identify the site occupied by the ligand. The mechanism of modulation may originate from a change in properties of the CRP subunit or inter-subunit communication or both. Thus, the binding affinity of cyclic nucleotides to both monomeric and dimeric CRP will be determined. There is indirect evidence to indicate a change in protein dynamics as CRP assumes the various functional states. Hence, the effect of mutation on protein dynamics will be monitored by hydrogen exchange using FT-IR and mass spectrometry. Modulation may be linked to the asymmetric nature of the bent gal DNA. The role of structural asymmetry in CRP-DNA interaction will be investigated by cross linking and in vitro transcription assays. The research program is a comprehensive biophysical study on modulation in DNA recognition.
