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首页> 外文学位 >H. pylori NikR: A New Nickel Regulatory Protein.
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H. pylori NikR: A New Nickel Regulatory Protein.

机译：幽门螺杆菌NikR：一种新的镍调节蛋白。

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摘要

My research has focused on understanding the mechanism of DNA recognition by the nickel regulatory protein NikR from Helicobacter pylori (HpNikR). H. pylori colonize the highly acidic gastric epithelium of the human stomach. One feature that enables H. pylori to survive under acidic conditions is the ability to release large quantities of ammonia, produced by the NikR regulated enzyme urease, to neutralize its immediate environment. HpNikR also regulates the expression of multiple other genes as either an activator or repressor including those involved in nickel ion homeostasis, acid adaptation and iron uptake. The genes for which direct regulation have been established contain variable recognition sequences; and the biophysical basis for DNA recognition and discrimination by HpNikR is currently unresolved. Crystallographic studies produced an unanticipated structure of Holo-HpNikR in which nickel ions are coordinated to two distinct binding sites: a 4-coordinate, square planar site (called the 4-site) and a 5/6-coordinate square-pyramidal/octahedral site (called the 5/6-site) in contrast to previous NikR structures where all four ions are coordinated to square planar sites. A mutant of HpNikR called H74A was designed to force all four nickels to the 4-sites, and the crystal structure confirmed singular coordination. DNA binding studies revealed that when Ni(II) is restricted from the 5/6 sites, DNA binding properties are abrogated compared to Holo-HpNikR. These data support a mechanism in which nickel coordination to the 5/6 site of HpNikR is critical for function. A reporter assay was also developed to monitor transcription of urease as a function of nickel concentration and promoter sequence directly within H. pylori. Unexpectedly, initial findings from this study directed us towards a novel cooperative effect in vivo where the presence of active HpArsR-P, from the ArsRS two component system, in conjunction with HpNikR leads to maximum Ni(II) dependent induction of urease. HpArsR is also able to bind the HpNikR PureA mutant operator sites in vitro as determined via fluorescence anisotropy at neutral pH with a high nanomolar to low micromolar affinity. Through the use of both in vitro and in vivo approaches, a novel model has been proposed for DNA recognition by HpNikR.
机译：我的研究集中在了解幽门螺杆菌（HpNikR）的镍调节蛋白NikR对DNA识别的机制。幽门螺杆菌定植在人胃的高酸性胃上皮中。使幽门螺杆菌能够在酸性条件下生存的一项功能是能够释放由NikR调节的酶脲酶产生的大量氨，以中和其直接环境。 HpNikR还调节多种其他基因的表达，使其作为激活物或阻遏物，包括那些与镍离子稳态，酸适应和铁吸收有关的基因。已建立直接调控的基因包含可变识别序列； HpNikR识别和识别DNA的生物物理基础目前尚未解决。晶体学研究产生了Holo-HpNikR的意外结构，其中镍离子与两个不同的结合位点配位：一个4坐标的方形平面位点（称为4位）和一个5/6坐标的方形金字塔形/八面体位点（称为5/6位）与以前的NikR结构相反，在该结构中，所有四个离子都配位到方形平面位。设计了一个名为H74A的HpNikR突变体，将所有四个镍都强迫到4位，并且晶体结构证实了奇异的配位。 DNA结合研究表明，当Ni（II）限制在5/6位时，与Holo-HpNikR相比，DNA结合特性被取消。这些数据支持了一种机制，其中镍与HpNikR的5/6位点的配位对于功能至关重要。还开发了一种报告基因测定法，以直接在幽门螺杆菌中监测尿素酶的转录与镍浓度和启动子序列的关系。出乎意料的是，这项研究的初步发现将我们引向了一种新的体内协同效应，其中来自ArsRS两组分系统的活性HpArsR-P与HpNikR结合导致尿素酶的最大Ni（II）依赖性诱导。 HpArsR还能够在体外结合HpNikR PureA突变体操作位点，该位点通过中性pH值下的荧光各向异性确定，具有高纳摩尔至低微摩尔亲和力。通过使用体外和体内方法，已提出了一种新的模型用于HpNikR的DNA识别。

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  • 作者

    West, Abby L.;

  • 作者单位

    University of Maryland, Baltimore.;

  • 授予单位 University of Maryland, Baltimore.;
  • 学科 Chemistry Biochemistry.;Biology Microbiology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 265 p.
  • 总页数 265
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 地球物理学;
  • 关键词

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