STRUCTURAL STUDIES OF RED CELL FERRITINS

铁蛋白是一种多亚基蛋白， 在动物、植物和动物的中空蛋白质外壳中， 和细菌细胞。 动物体内至少有三种铁蛋白基因 在不同的组织中表达不同， 环境条件和发展;其速度和机制， 铁的积累、储存和周转也不同。 我们的目标是 研究铁蛋白的作用机制和结构 这些功能差异的基础上，通过使用X射线晶体学， 确定并比较重组体的三维结构 两栖动物红色H和L铁蛋白以及一组具有 在不同的实验条件下改变功能。 特别 重点将放在消除或恢复能力的突变体上 形成早期的铁-酪氨酸中间体，该中间体仅在H- 铁蛋白，涉及一组与铁蛋白相关的移动的侧链的突变， 我们在之前的研究中已经发现了一种水合作用， 对称轴附近的突变。 我们还将使用在 这个实验室专门用于模拟大分子组装 来模拟和比较各种结构中的静电效应。 的 将确定各种蛋白质的功能特性， 在溶液中进行比较，主要通过EXAFS，共振拉曼，穆斯堡尔和 紫外-可见吸收光谱。 这项建议的基础是我们的 野生型两栖动物红细胞L-铁蛋白和两种突变体的早期研究 这表明铁蛋白的结构比 先前已经认识到并且可以被差异溶剂化。 我们希望 阐明支配这些结构变化的规则及其 与功能的关系，特别是L-和H-的差异 铁蛋白 由于铁蛋白的亚基是四螺旋束， 这项研究将深入了解这种常见的结构基序是如何在 蛋白质适应序列和外部条件的变化。 的 铁蛋白的作用机制与理解铁蛋白的分子机制有关。 铁代谢的基础在正常健康和疾病， 地中海贫血、血色素沉着症和镰状细胞性贫血或其他病症 需要大量输血或血液透析。

Ferritin is a multisubunit protein that concentrates and sequesters as many as 4,500 iron atoms within a hollow protein shell in animal, plant and bacterial cells. There are at least three ferritin genes in animals that differ in expression in different tissues, under different environment conditions and with development; their rates and mechanisms of iron accumulation, storage and turnover are also different. Our goal is to investigate the mechanism of action of ferritin and the structural basis of these functional differences by using X-ray crystallography to determine and compare the three-dimensional structures of recombinant amphibian red H and L ferritins and a set of single site mutants with altered function under different experimental conditions. Particular emphasis will be placed on mutants that eliminate or restore the ability to form the early iron-tyrosine intermediate that forms only in H- ferritin, mutations involving a set of mobile sidechains associated with a spine of hydration that we have identified in previous studies and mutations near the symmetry axes. We will also use a package developed in this laboratory specifically for modeling large macromolecular assemblies to model and compare electrostatic effects in the various structures. The functional properties of the various proteins will be determined and compared in solution, principally by EXAFS, resonance Raman, Mossbauer and uv-visible absorption spectroscopy. The basis of this proposal is our earlier studies of wild type amphibian red cell L-ferritin and two mutants which indicated that the ferritin structure is more plastic than had previously been appreciated and can be differentially solvated. We wish to elucidate the rules that govern these structural changes and their relationship to function, particularly the differences in L- and H- ferritins. Since the subunits of ferritin are four-helix bundles, this study will provide insight into how this common structural motif in proteins accommodates to changes in sequence and external conditions. The mechanism of action of ferritin is relevant to understanding the molecular basis of iron metabolism in normal health and in diseases such as thalassemia, hemochromatosis and sickle cell anemia or other conditions requiring hypertransfusion or hemodialysis.