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.
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