X-RAY STRUCTURAL STUDIES OF LACTOFERRIN

这项研究的主要目的有两个：(一)确定 乳铁蛋白的生物学作用的分子基础 牛奶、其他体液和白细胞中的铁结合蛋白，以及 (Ii)将乳铁蛋白的研究成果推广到更广泛的领域 转铁蛋白家族及其结合和释放机制 通常是蛋白质。 这项研究将使用X射线的补充技术 结晶学和定点突变来确定哪些因素 确定金属和阴离子的结合亲和力和结合机理 放手。这将对理解对 生物体液中铁和其他金属的水平，身体防御 机制(特别是抗菌活性)，生物学方面 母乳和婴儿健康，以及微量元素的生物可利用性。 乳铁蛋白的X射线结构研究结果 将被用来选择突变的目标，突变的乳铁蛋白 将克隆的cDNA在BHK细胞中表达，并 通过X射线结晶学和溶液研究进行了分析。具体目标 包括： (I)重组N-叶的高分辨X射线结构分析 人乳铁蛋白，有铁结合和无铁两种形式。 (Ii)通过诱变研究金属和阴离子结合决定因素 Asp 60、Arg 121和两个Tyr配体，以及结晶和 突变体的结构分析。这也将解决控制 金属专一性和蛋白质中金属位置的设计。 (Iii)分析转铁蛋白的构象变化 乳铁蛋白的铰链区。(四)潜在受体的研究 通过特定残基的突变形成结合区。 (V)糖基化的结构和功能效应分析 通过突变适当的ASN残基突变乳铁蛋白。 (Vi)利用乳铁蛋白区域构建嵌合分子 和转铁蛋白，以探讨两者结构和功能的差异 两个人。嵌合分子将被结晶并通过 X射线结晶学。 (Vii)天然转铁蛋白变体的X射线结构分析， 牛乳铁蛋白与肿瘤相关蛋白 黑素转铁蛋白。

The broad aims of this research are twofold, (i) to determine the molecular basis for the biological roles of lactoferrin, the major iron-binding protein in milk, other bodily secretions and leukocytes, and (ii) to extend the results from the research on lactoferrin to the wider transferrin family and to mechanisms of binding and release by binding proteins generally. The research will use the complementary techniques of X-ray crystallography and site-directed mutagenesis to define the factors which determine metal and anion binding affinity and mechanisms of binding and release. It will have implications for understanding the control of levels of iron and other metals in biological fluids, bodily defence mechanisms (especially antibacterial activity), aspects of the biology of human milk and infant health, and the bioavailability of trace elements. The results from our previous X-ray structural studies on lactoferrin will be used to select targets for mutagenesis, and mutant lactoferrins will be obtained by expression of the cloned cDNA in BHK cells and analyzed by X-ray crystallographic and solution studies. Specific aims are: (i) High resolution X-ray structure analyses of the recombinant N-lobe of human lactoferrin, in both iron-bound and iron-free forms. (ii) Investigation of metal and anion binding determinants by mutagenesis of Asp 60, Arg 121 and the two Tyr ligands, and crystallization and structure analysis of mutants. This will also address the control of metal specificity and the design of metal sites in proteins generally. (iii) Analysis of conformational change in transferrins by mutations in the hinge region of lactoferrin. (iv) Investigation of potential receptor binding regions by mutagenesis of specific residues. (v) Analysis of the structural and functional effects of glycosylation of lactoferrin by mutation of the appropriate Asn residues. (vi) Construction of chimeric molecules using regions from lactoferrin and transferrin, to probe structural and functional differences between the two. The chimeric molecules will be crystallized and analyzed by X-ray crystallography. (vii) X-ray structure analyses of natural transferrin variants, in particular bovine lactoferrin and the tumor-associated protein melanotransferrin.