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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 几乎所有的生物体都需要铁来维持正常的代谢功能。但由于其 由于铁与氧的反应性，高水平的铁可以产生活性氧物质，从而破坏DNA、脂质和蛋白质。在人类中，铁调节蛋白（IRP 1和IRP 2）协调铁稳态基因的转录后调节与细胞铁的可用性。当细胞铁浓度高时，IRP 1获得铁硫簇并催化柠檬酸盐和异柠檬酸盐的相互转化。相反，当细胞铁浓度低时，IRP 1调节铁稳态基因的翻译。这些功能变化需要IRP 1进行结构重组，从而两个结构域要么彼此旋转以形成活性位点，要么彼此旋转远离以形成RNA结合裂缝。apo-IRP 1的构象和IRP 1的结构成分，促进刚体域旋转仍然未知。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IRP 1的全局构象。为了验证这一假设，在铰链区和接头区实施了旨在防止IRP 1结构转变的突变。初步的功能分析表明，铰链突变体的酶活性降低了90%，而RNA结合亲和力与野生型相似。为了进一步了解这些突变如何影响全局IRP 1构象，我们提出了小角度X射线散射实验，比较旋转半径，对分布函数和低分辨率的分子包膜的野生型IRP 1铰链和接头突变体。

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.