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.
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