STRUCTURAL STUDIES OF RED CELL FERRITINS

铁蛋白是一种多亚单位蛋白质，它浓缩和隔离为 在动物、植物的中空蛋白质壳内有多达4500个铁原子 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的不同，在不同的 环境条件与发展；它们的比率和机制 铁的积累、储存和周转也不同。我们的目标是 探讨铁蛋白的作用机制及结构 通过使用X射线结晶学来研究这些功能差异的基础 重组蛋白三维结构的测定与比较 两栖动物红H和L铁蛋白和一组具有 不同实验条件下的功能变化。特例 重点将放在消除或恢复这种能力的突变体上。 形成早期的铁-酪氨酸中间体，它只在H-中形成 铁蛋白，涉及一组与以下相关的移动侧链突变 我们在以前的研究中发现的一种水化脊椎 对称轴附近的突变。我们还将使用在 这个实验室专门用来模拟大分子组装 对各种结构中的静电效应进行建模和比较。这个 各种蛋白质的功能特性将被确定并 在溶液中进行了比较，主要是用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 野生型两栖类红细胞L铁蛋白及其两个突变体的早期研究 这表明铁蛋白结构比以前更具可塑性。 以前是被理解的，并且可以被差异化地解决。我们希望 阐明支配这些结构性变化的规则及其 与功能的关系，特别是L-和H-的区别 铁素体。由于铁蛋白的亚基是四股螺旋束，所以 研究将深入了解这一常见的结构主题是如何 蛋白质能适应序列和外部条件的变化。这个 铁蛋白的作用机制与分子水平的理解有关 正常健康和疾病中铁代谢的基础 地中海贫血、血色沉着症、镰状细胞性贫血或其他情况 需要过度输血或血液透析的。

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.