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Biology facilitated by heme proteins as seen in Cimex nitrophorin and ecdysone inducible protein 75.

机译：血红素蛋白促进了生物学的发展，如Cimex nitrophorin和蜕皮激素可诱导蛋白75所示。

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摘要

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.;Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of the proteins using UV-visible spectroscopy indicates that E75 has a b type heme with a low spin six-coordinate ferric iron. In the E75 heme binding domain, the heme exhibits an unstable ferrous state and only binds NO and CO at high non-physiological levels. These data place into doubt the suggested roles for E75 as a gas regulated transcription regulator.
机译：本文以两种血红素蛋白为例，对血红素如何促进生物学的研究。我分别介绍了我对Cimex nitrophorin的机理研究和对蜕皮激素可诱导蛋白75的初步研究。氮素是唾液中的血红素蛋白，被食血昆虫用来向受害者释放NO，从而导致血管舒张和止血。臭虫亚硝基荧光素cNP是一种硫醇盐血红素蛋白，它通过不寻常的血红素辅助S-亚硝化反应来实现，需要近端配体裂解。本文通过突变，晶体学和瞬态动力学方法探讨了这种机理。我介绍了两个NO结合事件的详细调查，一个在血红素上，另一个在近半胱氨酸上。血红素亚硝酰基显示出由于近端半胱氨酸配体的表观质子化而引起的明显的pH依赖性，这是cNP功能的关键特征。 cNP突变蛋白的结构和光谱表明，SNO修饰具有调节性。激光闪光光解测量和突变蛋白的结构揭示了位阻对SNO稳定性的负面影响。昆虫胚胎发生和变态的研究表明，蜕皮激素通过其靶标蜕皮激素受体发挥调节作用。蜕皮激素以时间和组织依赖性方式触发几种核受体的表达，进而进行基因调控。蜕皮激素诱导蛋白75（E75）是一种核受体，是早期蜕皮激素响应基因产物，调节归因于蜕皮激素的一部分发育活动。我们正在研究埃及伊蚊（Aedes aegypti）的E75，以揭示其在蚊子蜕皮激素信号传导中的作用。我已经使用杆状病毒驱动的表达在SF9细胞中表达并部分纯化了全长蛋白，并纯化了从通过在大肠杆菌中表达而获得的包涵体中可溶的血红素结合域，使其同质。使用紫外可见光谱对蛋白质进行的初步表征表明，E75具有b型血红素，具有低自旋六坐标三价铁。在E75血红素结合域中，血红素显示出不稳定的亚铁状态，仅以高非生理水平结合NO和CO。这些数据使人们怀疑E75作为气体调节转录调节因子的建议作用。

著录项

  • 作者

    Badgandi, Hemant.;

  • 作者单位

    The University of Arizona.;

  • 授予单位 The University of Arizona.;
  • 学科 Biology Entomology.;Biophysics General.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 156 p.
  • 总页数 156
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 昆虫学;生物化学;生物物理学;
  • 关键词

  • 入库时间 2022-08-17 11:38:23

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