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首页> 外文学位 >Biochemical characterization of CgrA, a novel cGMP-binding CRP homolog from Rhodospirillum centenum.
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Biochemical characterization of CgrA, a novel cGMP-binding CRP homolog from Rhodospirillum centenum.

机译：CgrA的生化特征，CgrA是一种来自百日红螺螺旋菌的新型cGMP结合CRP同源物。

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摘要

Bacterial physiological status is controlled in part by a signal transduction network of small molecules that integrate information from both their extracellular and intracellular environment of the cell. Several intracellular signaling pathways are controlled by cyclic nucleotides that integrate information from outside to and inside of the cell. Under proper circumstances, these small molecules elicit appropriate responses by acting as a signal to a receptor molecule that contains an output domain. The well-characterized family of proteins that sense and bind small nucleotide molecules is the E. coli CRP family of transcriptional regulators. In my doctoral thesis, I have characterized a novel CRP homolog, called CgrA that binds cyclic guanosine monophosphate (cGMP) as a signal to trigger Rhodospirillum centenum to induce metabolically dormant cysts that survive harsh environmental conditions. In this thesis, I have utilized a combination of biochemical and biophysical studies to show that CgrA binds cGMP in a manner similar to how E. coli CRP binds cAMP. I also used fluorescence anisotropy to show that cGMP nucleotide binding by CgrA stimulates recognition of specific DNA sequences in a manner that is similar to that of CRP binding to DNA in the presence of cAMP. This work thus highlights and contrasts the mechanism of gene expression by R. centenum CgrA with that of the well characterized E. coli CRP. To obtain an understanding of how CgrA might perceive cGMP and differentiate it from other cyclic nucleotides, I also used a combination of homology modeling and site-directed mutagenesis using E. coli CRP-cAMP crystal structure as a reference molecule. Mutagenesis studies have identified Arg100 as an important residue in establishing hydrogen bond formation with the negative phosphate backbone of the ribose moiety of the cGMP and thus an Arg100Leu mutation completely abolishes binding to any cyclic nucleotide. Additional mutations however, failed to identify any amino acid residues that confer cGMP specificity over that of cAMP. Thus, although both proteins are homologues, they share remote similarity in their core ligand binding pocket suggesting that CgrA binds cGMP in a manner that is distinct from cAMP binding by CRP. I also undertook crystallization trials with full-length native CgrA that has yielded two promising crystal growth conditions one of which closely resembles the crystallization conditions of Clp, a CRP homolog from Xanthomonas campestris.
机译：细菌的生理状态部分受小分子的信号转导网络控制，这些信号转导网络整合了来自其细胞外和细胞内环境的信息。几种胞内信号传导途径受环状核苷酸控制，环状核苷酸整合了细胞外部和内部的信息。在适当的情况下，这些小分子通过充当包含输出域的受体分子的信号来引发适当的响应。感知并结合小核苷酸分子的蛋白质家族非常有名，它是转录调节因子E. coli CRP家族。在我的博士论文中，我描述了一种新颖的CRP同源物，称为CgrA，它与环状鸟苷单磷酸（cGMP）结合，作为触发百日红螺螺旋藻以诱导在恶劣环境条件下存活的代谢性休眠囊肿的信号。在这篇论文中，我利用生化和生物物理研究的结合来证明CgrA以类似于大肠杆菌CRP结合cAMP的方式结合cGMP。我还使用荧光各向异性来表明CgrA与cGMP核苷酸的结合刺激了特定DNA序列的识别，其方式类似于在cAMP存在下CRP与DNA结合的方式。因此，这项工作突出了并对比了百叶红球菌CgrA与特征明确的大肠杆菌CRP的基因表达机制。为了了解CgrA如何感知cGMP并将其与其他环状核苷酸区分开，我还使用了同源建模和以大肠杆菌CRP-cAMP晶体结构为参考分子的定点诱变的组合。诱变研究已确定Arg100是与cGMP核糖部分的负磷酸根骨架建立氢键形成的重要残基，因此Arg100Leu突变完全消除了与任何环状核苷酸的结合。然而，其他突变未能鉴定出赋予cGMP特异性高于cAMP的任何氨基酸残基。因此，尽管这两种蛋白都是同源物，但它们在其核心配体结合口袋中具有遥远的相似性，表明CgrA以不同于cAMP与CRP结合的方式结合cGMP。我还用全长天然CgrA进行了结晶试验，该试验产生了两个有希望的晶体生长条件，其中一个与Clp的结晶条件非常相似，Clp是来自Xanthomonas campestris的CRP同源物。

著录项

  • 作者

    Roy Chowdhury, Sugata.;

  • 作者单位

    Indiana University.;

  • 授予单位 Indiana University.;
  • 学科 Biology Microbiology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 206 p.
  • 总页数 206
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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