The Structure and Function of Human Serum Transferrin

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; 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 (