STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

大肠杆菌环AMP受体蛋白的研究

• 批准号: 3271225
• 负责人: JOSEPH S KRAKOW
• 金额: $ 15.47万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-04-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271225
• 关键词: DNA binding protein; Escherichia coli; bacterial genetics; binding proteins; conformation; crosslink; cyclic AMP; cyclic AMP receptors; gel electrophoresis; genetic transcription; laboratory mouse; monoclonal antibody; protein folding; protein structure function; radiotracer; site directed mutagenesis; temperature sensitive mutant

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.

cAMP-CRP的结构已被阐明。 生化和 CRP 和 cAMP-CRP 之间的免疫学差异已有描述。 在缺乏 cAMP 或低 cAMP 浓度下发挥作用的突变体 （CRP）已被隔离。 封闭构象是如何维持的 未配体的 CRP 以及 cAMP 的结合如何导致开放的形成 构象仍有待解决。 为了获得有关 CRP 的结构，我们将在 CRP 位点产生位点特异性突变 显示在 cAMP 存在的情况下可访问，但在未配体的情况下不可访问 状态。 这些位点已在我们的敏感性研究中定义 cAMP-CRP 的特定区域可被蛋白酶攻击或被抗-CRP 结合 CRP 单克隆抗体。 其他要突变的区域或位点是 推测 cAMP-CRP 晶体结构具有灵活性；这些地区是 存在于螺旋或β链之间的转圈和环中。 特别的 甘氨酸残基似乎在相关调节蛋白中是保守的 包括CRP。 甘氨酸残基可作为构象点 响应 cAMP 结合的旋转将按位点更改为丙氨酸 特异性突变。 切除C端7个氨基酸残基 羧肽酶 Y 导致 DNA 结合活性和能力丧失 支持 lac 转录。 每个中的氨基酸取代 通过位点特异性诱变制备通过 Lys-201 的残基。 突变体将允许确定哪些残基是 参与维持构象稳定性 参与 DNA 结合 和激活转录的能力。 最终的方法将涉及 使用含有 CRP 随机改变的长引物进行随机诱变 顺序。 在 CRP 突变体群体中，可能存在这样的突变体： 不结合 cAMP，不需要 cAMP (CRP)，结合 cAMP 但不能进行 cAMP 引起的构象变化，结合 cAMP 并经历 必要的构象变化并结合到启动子 DNA 位点，但不能 刺激转录（阳性对照突变体）和那些可能 折叠缺陷（温度敏感）。 可能的联系地点 CRP 和 RNA 聚合酶之间的相互作用将通过光交联进行探测。 这 抗CRP单克隆抗体表位的位置将是 决定。