The structure of cAMP-CRP has been elucidated. Biochemical and immunological differences between CRP and cAMP-CRP have been described. Mutants which function in the absence of cAMP or at low cAMP concentrations (CRP) have been isolated. How the closed conformation is maintained in unliganded CRP and how binding of cAMP results in the formation of the open conformation remain to be resolved. In order to gain information on the structure of CRP we will generate site specific mutations at CRP sites shown to be accessible in the presence of cAMP but not in the unliganded state. These sites have been defined in our studies on the sensitivity of particular regions of cAMP-CRP to attack by proteases or binding by anti- CRP monoclonal antibodies. Other regions or sites to be mutated are those inferred to be flexible in the cAMP-CRP crystal structure; such regions are present in turns and loops between helices or beta strands. Particular glycine residues appear to be conserved in related regulatory proteins including CRP. Glycine residues which may act as points for conformational rotation in response to binding of cAMP will be changed to alanine by site specific mutation. Excision of the C-terminal 7 amino acid residues by carboxypeptidase Y results in loss of DNA binding activity and the ability to support lac transcription. Amino acid substitutions in each of the residues through Lys-201 will be prepared by site specific mutagenesis. The mutants will allow for the determination of which residue(s) are involved in maintaining conformational stability involved in DNA binding and ability to activate transcription. The final approach will involve random mutagenesis using long primers containing random alterations in CRP sequence. Within the population of CRP mutants there may be those which do not bind cAMP, do not require cAMP (CRP), bind cAMP but cannot undergo the conformational changes elicited by cAMP, bind cAMP and undergo the requisite conformational changes and bind to promoter DNA sites but cannot stimulate transcription (positive control mutants) and those which may be defective in folding (temperature sensitive). The possible contact site between CRP and RNA polymerase will be probed by photocross-linking. The location of the epitopes for the anti-CRP monoclonal antibodies will be determined.
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