STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

CAMP-CRP的结构已经阐明。生化和 已描述了CRP和cAMP-CRP之间的免疫学差异。 在没有cAMP或在低cAMP浓度下起作用的突变体 (C反应蛋白)已被分离。如何在中保持闭合构象 未连接的C反应蛋白和cAMP结合如何导致开放的形成 构象尚待解决。为了获得有关的信息 C反应蛋白的结构我们将在C反应蛋白位点产生特定的突变 证明在有cAMP在场的情况下可以接近，但在无配体的情况下不能 州政府。这些部位已经在我们的研究中被定义为 CAMP-CRP的特定区域可被蛋白酶攻击或被抗- C反应蛋白单抗。其他要突变的区域或位置是那些 推测在cAMP-CRP晶体结构中是灵活的；这些区域是 在螺旋或β链之间轮流和环状存在。特例 甘氨酸残基似乎在相关的调节蛋白中是保守的 包括C反应蛋白。甘氨酸残基可以作为构象的点 反应cAMP结合的旋转将被逐个位点改变为丙氨酸 特定的突变。C-末端7个氨基酸残基的切除 羧基肽酶Y导致DNA结合活性和能力的丧失 以支持Lac转录。每种植物中的氨基酸替换 通过Lys-201的残基将通过定点突变来制备。 突变体将允许确定哪些残基(S)是 参与维持DNA结合的构象稳定性 以及激活转录的能力。最后一种方法将涉及 利用含有C反应蛋白随机改变的长引物进行随机诱变 序列。在C反应蛋白突变体的种群中，可能存在这样的突变 不绑定cAMP，不需要cAMP(CRP)，绑定cAMP但不能经受 CAMP引发的构象变化，结合cAMP和经历 必需的构象变化和与启动子DNA位点的结合但不能 刺激转录(阳性对照突变体)和那些可能 折叠有缺陷(对温度敏感)。可能的联系地点 CRP和RNA聚合酶之间的关系将通过光交联法进行探索。这个 抗CRP单抗的表位位置将为 下定决心。

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.