ANALYSIS OF IRON REGULATORY PROTEIN 1 RIGID BODY DOMAIN ROTATION BY SAXS

通过 SAXS 分析铁调节蛋白 1 刚体结构域旋转

• 批准号: 8361304
• 负责人: KARL W VOLZ
• 金额: $ 0.2万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361304
• 关键词: Active Sites; Affect; Affinity; Biophysics; Citrates; Cleaved cell; DNA Damage; Funding; Genes; Grant; Homeostasis; Human; Iron; Iron Regulatory Protein 1; Iron-Regulatory Proteins; Isocitrates; Lipids; Metabolic; Molecular; Molecular Conformation; Mutation; National Center for Research Resources; Organism; Oxygen; Post-Transcriptional Regulation; Principal Investigator; Proteins; RNA Binding; Radial; Reactive Oxygen Species; Research; Research Infrastructure; Resolution; Resources; Rotation; Source; Sulfur; Testing; Translations; United States National Institutes of Health; cost; design; enzyme activity; insight; isocitrate; mutant; prevent; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Nearly all organisms require iron for normal metabolic function. However, due to its reactivity with oxygen, high levels of iron can generate reactive oxygen species which damage DNA, lipids and proteins. In humans, iron regulatory proteins (IRP1 and IRP2) coordinate post-transcriptional regulation of iron homeostasis genes with cellular iron availability. When cellular iron concentration is high, IRP1 acquires an iron sulfur cluster and catalyzes the inter-conversion of citrate and isocitrate. Conversely, when cellular iron concentration is low, IRP1 regulates translation of iron homeostasis genes. These functional changes require IRP1 to undergo structural reorganization whereby two domains either rotate toward each other to form an active site or rotate away from each other to form a RNA binding cleft. The conformation of apo-IRP1 and the structural components of IRP1 that facilitate rigid body domain rotation remain unknown. We hypothesized that the linker and hinge regions determine the global conformation of IRP1 by acting as axes of rigid body domain rotation. To test this hypothesis, mutations designed to prevent structural transitions of IRP1 were implemented in the hinge and linker regions. Preliminary functional analysis indicated that the enzyme activity of hinge mutants decreased by 90% compared to wild type, whereas the RNA binding affinity was similar to wild type. In order to gain further insight toward how these mutations affect global IRP1 conformation, we proposed small angle x-ray scattering experiments to compare radii of gyration, pair distribution functions and low-resolution molecular envelopes of wild type IRP1 to the hinge and linker mutants.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 几乎所有的生物体都需要铁来维持正常的代谢功能。然而，由于其 与氧的反应，高水平的铁会产生活性氧物种，从而破坏DNA、脂质和蛋白质。在人类中，铁调节蛋白(IRP1和IRP2)协调铁稳态基因的转录后调节与细胞铁的可获得性。当细胞铁浓度较高时，Irp1获得一个铁硫簇，并催化柠檬酸和异柠檬酸的相互转化。相反，当细胞铁浓度较低时，Irp1调节铁稳态基因的翻译。这些功能变化要求Irp1经历结构重组，即两个结构域要么朝向彼此旋转形成活性部位，要么相互旋转远离形成RNA结合裂隙。Apo-irp1的构象和促进刚体结构域旋转的irp1的结构成分仍然未知。我们假设连接区和铰链区通过作为刚体结构域旋转的轴来决定Irp1的整体构象。为了验证这一假设，在铰链和连接区实施了旨在防止Irp1结构转变的突变。初步的功能分析表明，与野生型相比，铰链突变体的酶活性降低了90%，而与RNA的亲和力与野生型相似。为了进一步了解这些突变是如何影响整体Irp1构象的，我们提出了小角x射线散射实验，比较了野生型Irp1的旋转半径、对分布函数和低分辨率分子膜与铰链和连接子突变体的差异。