RUI: Structural and Functional Investigations of Ferritin Heteropolymers Using a Novel Expression System

大多数生物体都进化出了一种机制，以维持铁作为必需营养素和铁作为潜在毒素之间的平衡。 铁蛋白是一种多亚基的铁储存和解毒蛋白，在铁的生物管理中起着核心作用。 本项目的目的是利用一种新的表达系统表达和纯化重组异铁蛋白，并首次将重组异铁蛋白与从动物组织和器官中提取的天然异铁蛋白之间的结构-功能关系联系起来。 异铁蛋白是一种复杂的分子，具有精致的结构纳米结构，由两种不同的亚基类型组成，称为H和L。尽管它们在80多年前被发现，但从未对重组产生的异铁蛋白进行过研究，也没有任何研究小组能够在体内合成模拟天然异铁蛋白的铁蛋白。 了解各种异铁蛋白群体之间的功能差异，以及不同比例的H-和L-亚基如何影响异铁蛋白的生物化学和功能特性，对于了解哺乳动物中铁的生物加工至关重要。这项工作还将为本科生提供现代生物化学和生物物理技术的培训，以及解决铁稳态挑战性问题所需的实验室技能，并为未来学术界或工业界的STEM职业做好准备。在整个项目中，本科生将参与有关不同亚基组成的异铁蛋白的生物合成，纯化和表征的原始研究，使用一种新的表达系统，提供铁蛋白分子内H-和L-亚基的任何所需组合，模仿各种组织和器官中发现的天然异铁蛋白。 为了实现这些目标，将采用分子生物学技术、光吸收和荧光光谱、毛细管电泳、差示扫描量热法、穆斯堡尔光谱、磁力测定法以及高分辨率扫描和透射电子显微镜的组合。 在这个项目中概述的广泛调查应提供基本的见解异铁蛋白的结构-功能关系和铁代谢的动态，无论是在系统和细胞水平。这个奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

Most organisms have evolved mechanisms to maintain a balance between iron as an essential nutrient and iron as a potential toxin. Ferritins are multi-subunit iron storage and detoxification proteins that play a central role in the biological management of iron. The goal of this project is to express and purify recombinant isoferritins using a novel expression system, and for the first time, correlate structure-function relationships between recombinant isoferritins and native isoferritins extracted from animal tissues and organs. Isoferritins are complex molecules with exquisite architectural nanostructures made up of two distinct subunit types, named H and L. Despite their discovery more than 8 decades ago, no studies have ever been performed on recombinantly produced isoferritins, nor has any research group been able to synthesize ferritins that mimic native isoferritins in-vivo. Understanding the functional differences between various isoferritin populations, and how different proportions of H- and L-subunits affect the biochemical and functional properties of isoferritins is of critical importance to understanding the biological processing of iron in mammals. This work will also provide undergraduate students training in modern biochemical and biophysical techniques, and laboratory skills necessary to address challenging questions about iron homeostasis, and preparation for future STEM careers in academia or industries. Throughout this project, undergraduate students will be involved in original research pertaining to the biosynthesis, purification, and characterizing of isoferritins of different subunits composition, using a novel expression system that provides any desired combinations of H- and L-subunits within the ferritin molecule, mimicking native isoferritins found in various tissues and organs. To achieve these goals, a combination of molecular biology techniques, light absorbance and fluorescence spectroscopy, capillary electrophoresis, differential scanning calorimetry, Mossbauer spectroscopy, magnetometry, and high-resolution scanning and transmission electron microscopy will be employed. The extensive investigation outlined in this project should provide fundamental insights into isoferritins structure-function relationships and the dynamics of iron metabolism, both at the systemic and the cellular levels.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.