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STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

大肠杆菌环AMP受体蛋白的研究

基本信息

批准号：
2173953
负责人：
JOSEPH S KRAKOW
金额：
$ 15.21万
依托单位：
HUNTER COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1976
资助国家：
美国
起止时间：
1976-04-01 至 1995-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2173953
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 nucleic acid sequence protein folding protein structure function radiotracer site directed mutagenesis temperature sensitive mutant

项目摘要

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.

已阐明cAMP-CRP的结构。生化和已经描述了CRP和cAMP-CRP之间的免疫学差异。在不存在cAMP或低cAMP浓度下起作用的突变体 (CRP)被隔离了封闭构象是如何在未配体的CRP以及cAMP的结合如何导致开放的构象仍有待解决。为了获得关于我们将在CRP位点产生位点特异性突变显示在cAMP存在下可接近，但在未配体的状态这些位点已经在我们的研究中定义， cAMP-CRP的特定区域被蛋白酶攻击或被抗- CRP单克隆抗体。待突变的其他区域或位点是那些推测在cAMP-CRP晶体结构中是柔性的;这样的区域是存在于螺旋或β链之间的圈和环中。特别甘氨酸残基似乎在相关的调节蛋白中是保守的包括CRP。甘氨酸残基，其可作为构象的点响应cAMP结合的旋转将通过位点改变为丙氨酸特殊突变 C-末端7个氨基酸残基的切除，羧肽酶Y导致DNA结合活性的丧失，以支持lac转录。在每一个氨基酸序列中的氨基酸取代将通过位点特异性诱变来制备通过Lys-201的氨基酸残基。突变体将允许确定哪些残基是参与维持DNA结合的构象稳定性和激活转录的能力。最后的方法将涉及使用含有CRP随机改变的长引物的随机诱变顺序在CRP突变体的群体中，可能存在这样的突变体。不结合cAMP，不需要cAMP（CRP），结合cAMP但不能接受由cAMP引起的构象变化，结合cAMP并经历必需的构象变化，并结合到启动子DNA位点，但不能刺激转录（阳性对照突变体）和那些可能折叠不良（对温度敏感）。可能的接触点将通过光交联探测CRP和RNA聚合酶之间的相互作用。的抗CRP单克隆抗体的表位的位置将是测定