X RAY STRUCTURAL STUDIES OF LACTOFERRIN

描述(摘自申请人摘要)：乳铁蛋白是一种 来自母乳、其他分泌物和白细胞的铁结合蛋白和 具有广泛的已建立或提议的生物学活动。 它也是与血清蛋白转铁蛋白密切相关的一部分 一组蛋白质，调节铁的水平，可能还有其他 金属，在动物的体液中。这项研究的主要目的是 (I)确定人类生物学活动的分子基础 乳铁蛋白，(Ii)通过以下方式解决铁稳态的更广泛问题 将这些结构-功能研究扩展到血清转铁蛋白，以及(Iii) 研究这些蛋白质特异性的结构基础。它 将对理解铁和铁的水平的控制产生影响 与铁病有关的体液中的其他微量元素 超负荷或不足；身体防御机制，尤指抗菌 和抗氧化活性；母乳和婴儿的生物学方面 健康和微量元素的生物可利用性。 这项研究将使用x射线结晶学的补充技术。 和定点突变来定义决定金属和 阴离子结合亲和力和结合与释放的机理，并以此为探针 转铁蛋白结合和运输的多功能性。这是一个合乎逻辑的 乳铁蛋白和乳铁蛋白的结晶学研究 新开发的转铁蛋白结构工作；结构知识将是 用于选择突变的目标，突变的蛋白质将被 用X射线结晶学和溶液研究对其进行了表达和表征。 具体目标是：1.利用诱变和结晶学分析 铁位置后面的碱性残留物在影响铁中的重要性 放手。2.分析铁配体和阴离子结合的作用 在测定金属的阴离子亲和力和特异度方面。3.至 用X射线确定转铁蛋白中协同作用的结构基础 具有不同构象状态和不同构象的分子分析 两个肺叶的铁质状态。4.探讨乳铁蛋白的拟议作用 经结晶和X-射线结构分析确定为转录因子 乳铁蛋白-DNA复合体。5.调查绑定的多功能性 通过转铁蛋白，通过Ru(III)抗肿瘤络合物的结晶 据报道由转铁蛋白携带的药物，以及通过对 一种新型的转铁蛋白，它能结合一种有效的有机毒素来代替铁。6. 完成糖基化对结构和结构影响的研究 转铁蛋白的功能。

DESCRIPTION (Adapted from applicant's abstract): Lactoferrin is a prominent iron-binding protein from human milk, other secretions, and leukocytes and possesses a wide variety of established or proposed biological activities. It is also, with the closely-related serum protein transferrin, part of the team of proteins that regulates the levels of iron, and possibly other metals, in the body fluids of animals. The broad aims of this research are (i) to determine the molecular basis for the biological activities of human lactoferrin, (ii) to address the wider questions of iron homeostasis by extending these structure-function studies to serum transferrin, and (iii) to investigate the structural basis of specificity in these proteins. It will have implications for understanding the control of levels of iron and other trace elements in body fluids, with relevance to diseases of iron overload or deficiency; bodily defense mechanism, especially antibacterial and antioxidant activity; aspects of the biology of human milk and infant health, and the bioavailability of trace elements. The research will use the complementary techniques of x-ray crystallography and site-directed mutagenesis to define the factors that determine metal and anion binding affinity and mechanisms of binding and release, and to probe the versatility of binding and transport by transferrins. It is a logical extension of our previous crystallographic studies of lactoferrin and newly-developed structural work on transferrin; structural knowledge will be used to select targets for mutagenesis and the mutant proteins will be expressed and characterized by x-ray crystallography and solution studies. Specific aims are: 1. To use mutagenesis and crystallography to analyze the importance of basic residues behind the iron site in influencing iron release. 2. To analyze the roles of the iron ligands and anion binding groups in determining metal an anion affinity and specificity. 3. To determine the structural basis of cooperativity in transferrins by x-ray analysis of molecules with different conformational states and different iron status in the two lobes. 4. To probe the proposed role of lactoferrin as a transcription factor by crystallization and x-ray structural analysis of lactoferrin-DNA complexes. 5. To investigate the versatility of binding by transferrins, by crystallization of complexes with Ru(III) anti-tumor drugs that are reportedly carried by transferrins, and by x-ray analysis of a novel transferrin that binds a potent organic toxin in place of iron. 6. To complete a study of the effects of glycosylation on the structure and function of transferrin.