X-RAY STRUCTURAL STUDIES OF LACTOFERRIN

这项研究的主要目的有两个方面，（一）确定 乳铁蛋白的生物学作用的分子基础， 牛奶、其他身体分泌物和白细胞中的铁结合蛋白，以及 (ii)将乳铁蛋白的研究成果扩展到更广泛的领域， 转铁蛋白家族以及结合和结合释放机制 蛋白质一般。 这项研究将使用X射线的补充技术 晶体学和定点诱变来确定 确定金属和阴离子结合亲和力和结合机制， release. 这将对理解控制 生物体液中的铁和其他金属含量，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利用度。 我们以前对乳铁蛋白的X射线结构研究的结果 将用于选择诱变的靶点， 将通过在BHK细胞中表达克隆的cDNA获得， 通过X射线晶体学和溶液研究分析。 具体目标 为： (i)重组人大肠杆菌N叶的高分辨X射线结构分析 人乳铁蛋白，铁结合和无铁形式。 (ii)金属和阴离子结合决定簇的诱变研究 的Asp 60，Arg 121和两个Tyr配体，和结晶， 突变体结构分析。 这也将解决控制 金属特异性和蛋白质中金属位点的设计。 (iii)转铁蛋白构象变化的突变分析 乳铁蛋白的铰链区。(iv)潜在受体研究 通过特异性残基的诱变结合区域。 (v)糖基化的结构和功能效应分析 通过适当的Asn残基的突变来产生乳铁蛋白。 (vi)使用来自乳铁蛋白的区域构建嵌合分子 和转铁蛋白，以探测 the two. 嵌合分子将被结晶并通过 X射线晶体学 (vii)天然转铁蛋白变体的X射线结构分析， 特别是牛乳铁蛋白和肿瘤相关蛋白 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.