The Structure and Function of Human Serum Transferrin

;​R o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o4 @F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT marcia steinberg marcia steinberg @ = 19：@ mv e = e " " " " " " " \ \ \ \ \ t ~ \ # c# Õ u f t 4 # “ # ”6 D " " " " 4 9505824 Ikeda Transferrin是一种由679个氨基酸（80000 MW）组成的单体糖蛋白，折叠成两个同源结构域。它是负责溶解和运输循环铁的蛋白质。在转铁蛋白的N端区域有一个铁结合位点，在C端区域有一个铁结合位点。在这两个畴中，结合的铁原子由六个配体控制。四种配体（一种天冬氨酸、两种酪氨酸和一种组氨酸）由蛋白质提供，两种配体由协同结合的碳酸盐阴离子提供。协同阴离子是铁结合所必需的，并且已经假设碳酸盐通过保守的精氨酸特异性地锚定在转铁蛋白两个结构域的铁结合间隙内。虽然转铁蛋白结合间隙中的铁配体是相同的，两个结构域的序列和结构也几乎相同，但N端结构域和C端结构域的热稳定性和铁结合性能不同。据推测，两个转铁蛋白结构域的热稳定性差异是由两个转铁蛋白叶的一个或两个组成亚结构的热稳定性差异引起的，而转铁蛋白N端和C端结构域的铁结合特性差异归因于两个结构域的铁结合间隙上氨基酸的差异。本研究的目的是检验这些假设。具体而言：1)将在毕赤酵母（酵母）中表达N端和C端半转铁蛋白，并确认重组N端和C端半转铁蛋白的总体完整性和活性。这将证明重组半转铁蛋白是一种快速、简便的制备方法。2)通过交换N端和C端半转铁蛋白的亚结构域产生杂交半转铁蛋白，利用重组蛋白来确定N端和C端半转铁蛋白的热稳定性和铁结合活性的差异是由半转铁蛋白的一个或两个组成亚结构引起的。3)构建半转铁蛋白突变体。这些突变体将被用来验证这样的假设，即Arg124和Arg456参与了协同阴离子的结合，使得这些氨基酸对铁与N和C端半转铁蛋白的结合至关重要。这些实验将首次比较和对比转铁蛋白两个结构域的相似突变。这将增加我们对铁与转铁蛋白结合机制的认识，并将帮助我们了解转铁蛋白如何起溶解铁的作用。铁是血液生成和细胞生长所必需的。简而言之，它是人类生活必不可少的；然而，铁在生理溶液中几乎不溶。因此，它必须被溶解，以便有足够的铁可供增殖细胞使用。在人类中，负责结合、溶解和运输循环铁的蛋白质是人血清转铁蛋白。在结构上，转铁蛋白是一种由679个氨基酸组成的蛋白质，折叠成两个结构域。一个结构域由氨基酸1到331组成，而几乎相同的第二个结构域由氨基酸339到679组成。转铁蛋白的每个结构域都有一个铁结合位点，虽然两个结构域的序列和结构几乎相同，但两个结构域的热稳定性和铁结合性能不同。该项目的目标是利用重组DNA技术分别产生转铁蛋白的每个结构域，并研究转铁蛋白的分离结构域，以确定为什么几乎相同的结构域表现出不同的铁结合特性和不同的热稳定性。这些研究将帮助我们了解转铁蛋白是如何结合铁的，以及转铁蛋白的铁结合特性是如何允许蛋白质溶解和运输铁的。*** @ ....()()))()() ;如果我是你，我是你，我是你，我是你，我是你。

; R o o t E n t r y F VEcI @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l VEcI VEcI 4 @ F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ =1 9: @ mv e = e " " " " " " " \ \ \ \ \ t ~ \ # C # Õ u f T 4 # " # " " " " 6 D " " " " 4 9505824 Ikeda Transferrin is a monomeric glycoprotein of 679 amino acids ( 80,000 MW) that is folded into two homologous domains. It is the protein responsible for solubilizing and transporting circulating iron. There is one iron binding site in the N terminal domain of transferrin and one iron binding site in the C terminal domain. In both domains the bound iron atom is held by six ligands. Four ligands (an aspartic acid, two tyrosines, and a histidine) are provided by the protein and two ligands are provided by a synergistically bound carbonate anion. The synergistic anion is required for iron binding and it has been hypothesized that the carbonate is specifically anchored within the iron binding clefts of both domains o f transferrin by a conserved arginine. Although the iron ligands in the binding clefts of transferrin are identical and the sequence and structure of the two domains are nearly identical, the thermal stability and iron binding properties of the N and C terminal domains differ. It has been hypothesized that the differences in the thermal stabilities of the two domains of transferrin are caused by differences in the thermal stabilities of one or both of the component substructures of the two transferrin lobes, while the differences in the iron binding properties of the N and C terminal domains of transferrin have been attributed to differences in the amino acids that line the iron binding clefts of the two domains. The goals of this study are to test these hypotheses. Specifically: 1 ) N and C terminal half transferrin will be expressed in Pichia pastoris (yeast) and the general integrity and activity of the recombinant N terminal and C terminal half transferrins will be confirmed. This will prove that recombinant half transferrins can be produced quickly and easily. 2) Hybrid half transferrins will be produced by exchanging the subdomains of N and C terminal half transferrin, and the recombinant proteins will be used to determine whether the differences in the thermal stability and iron binding activity of N and C terminal half transferrin are caused by one or both of the component substructures of the half transferrins. 3) Half transferrin mutants will be constructed. The mutants will then be used to begin to test the hypothesis that the involvement of Arg124 and Arg456 in the binding of the synergistic anion makes these amino acids essential for iron binding by N and C terminal half transferrin. These experiments will be the first to compare and contrast similar mutations in both domains of transferrin. This should increase our knowledge of the mechanisms of iron binding by transferrin and will help us to understand how transferrin functions to solubilize iron. %%% Iron is essential for blood production and cell growth. In short, it is essential for human life; however, iron is almost insoluble in physiological solutions. Consequently, it must be solubilized so that enough iron is available for proliferating cells. In humans, the protein that is responsible for binding, solubilizing, and transporting circulating iron is human serum transferrin. Structurally, transferrin is a 679 amino acid protein that is folded into two domains. One domain is formed by amino acids 1 to 331, while the nearly identical second domain is formed by amino acids 339 to 679. There is one iron binding site in each domain of transferrin, and although the sequence and structure of the two domains are nearly identical, the thermal stability and iron binding properties of the two domains of transferrin differ. The goals of this project are to use recombinant DNA technology to separately produce each domain of transferrin, and to study the separated domains of transferrin to determine why the nearly identical domains exhibit different iron binding characteristics and different thermal stabilities. These studies will help us to understand how transferrin binds iron, and how the iron binding characteristics of transferrin allow the protein to solubilize and transport iron. *** @ ....()()))()() ; S u m m a r y I n f o r m a t i o n (